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THE 


CHEMICAL    BASIS 


ANIMAL   BODY. 


THE 


CHEMICAL    BASIS 


OF    THE 


ANIMAL    BODY. 


^n  ^ppmtiix  to  JFostcr's  Kcxi-Mook  of  Pijgsiolosg, 

(Sixth  Edition.) 


BY 


A.  SHERIDAN   LEA,  MA.,  D.Sc,  P.R.S., 

UNIVERSITY  LECTITRER  IN  PHTSIOLOGY  IN  THE  UNIVERSITY  OF  CAMBRIDGE;    FELLOW  OF 
CAIUS   COLLEGE,    CAMBRIDGE. 


Nebj  fork: 

MACMILLAN    AND    CO. 

AND    LONDON. 
1893. 

[All  rights  reserved.'} 


Copyright,  1893, 
By  Macmillan  and  Co. 


SSnitorrsttg  ISrtss: 
John  Wilson  and  Son,  Cambridge,  U.S.A. 


PREFACE. 


rr^HE  following  Appendix  has  been  written  upon  the  same 
-*-  lines  as  in  former  editions,  save  that  it  has  been  enlarged, 
and  in  reality  now  constitutes  a  treatise  on  the  chemical  sub- 
stances occurring  in  the  animal  body.  As  in  former  editions 
it  is  entirely  the  work  of  Dr.  A.  Sheridan  Lea. 

The  references  given,  though  extensive,  are  not  intended  to 
be  exhaustive.  An  effort  has  been  made  to  make  the  refer- 
ences to  recent  work  as  complete  as  possible ;  other  references 
are  to  papers  which  themselves  give  full  references  and  will 
therefore  serve  as  a  guide  to  the  literature  of  the  subject ; 
and  some  have  been  inserted  in  order  to  inform  the  student 
of  the  dates  at  which  important  results  were  first  described. 

We  desire  to  express  our  thanks  to  Messrs.  Winter  of 
Heidelberg  for  the  six  figures  which  have  been  taken  from 
Krukenberg's  Grrimdriss  der  mediciniscJi-chemisehen  Analyse, 
and  to  Professor  Klihne  for  the  large  number  which  have  been 
taken  from  his  Lekrbucli  der  physiologischen  Chemie  (1868). 
A  few  have  been  drawn  in  wood  from  the  plates  in  Funke's 
Atlas  der  physiologischen   Chemie  (1858). 

We  are  also  indebted  to  Dr.  S.  Ruhemann  for  reading  the 
proofs  from  page  91  to  page  216,  in  which  the  text  contains 
many  formulae,  and  involves  special  chemical  knowledge. 

The  volume  is  paged  separately  from  the  rest  of  the  Text- 
JBook,  and  has  an  index  of  its  own.  Indeed  it  may  be  regarded 
as  an  independent  work.  The  references  to  the  body  of  the 
Text-Book  are  given  in  paragraphs. 

M.  FOSTER. 

A.   SHERIDAN   LEA. 

July,  1892. 


PART  v.- APPENDIX. 

THE    CHEMICAL    BASIS    OF    THE 
ANIMAL    BODY 

BY 

A.   SHERIDAN   LEA,  M.A,   Sc.D.,   F.RS., 

UNIVERSITY    LECTURER    IN    PHYSIOLOGY    IN   THE    UNIVERSITY    OF    CAMBRIDGE  J 
FELLOW   OF   CAIUS   COLLEGE,   CAMBRIDGE. 


APPENDIX. 

THE  CHEMICAL  BASIS  OF  THE  ANIMAL  BODY. 

The  animal  body,  from  a  chemical  point  of  view,  may  be 
regarded  as  a  mixture  of  various  representatives  of  three  large 
classes  of  chemical  substances,  viz.  proteids,  carbohydrates  and 
fats,  in  association  with  smaller  quantities  of  various  saline  and 
other  crystalline  bodies.  By  proteids  are  meant  bodies  contain- 
ing carbon,  oxygen,  hydrogen,  and  nitrogen  in  a  certain  proportion, 
varying  within  narrow  limits,  and  having  certain  general  features ; 
they  are  frequently  spoken  of  as  albuminoids.  By  carbohydrates 
are  meant  starches  and  sugars  and  their  allies.  We  have  also 
seen  that  the  animal  body  may  be  considered  as  made  up  on  the 
one  hand  of  actual  'living  substance,'  sometimes  spoken  of  as 
protoplasm  (see  §  5),  in  its  various  modifications,  and  on  the  other 
hand  of  numerous  lifeless  products  of  metabolic  activity.  We  do 
not  at  present  know  anything  definite  about  the  molecular  com- 
position of  the  active  living  substance ;  but  when  we  submit 
living  substance  to  chemical  analysis,  in  which  act  it  is  killed, 
we  always  obtain  from  it  a  considerable  quantity  of  the  material 
spoken  of  as  proteid.  And  many  authors  go  so  far  as  to  speak  of 
living  substance  or  protoplasm  as  being  purely  proteid  in  nature ; 
they  regard  the  living  protoplasm  as  proteid  material,  which  in 
passing  from  death  to  life  has  assumed  certain  characters  and 
presumably  has  been  changed  in  construction,  but  still  is  proteid 
matter ;  they  sometimes  speak  of  protoplasm  as  '  living  proteid ' 
or  '  living  albumin.'  It  is  worthy  of  notice,  however,  that  even 
simple  forms  of  living  matter,  like  that  constituting  the  body  of 
a  white  corpuscle,  forms  which  we  may  fairly  consider  as  the 
nearest  approach  to  native  protoplasm,  when  they  can  be  obtained 
in  sufficient  quantity  for  chemical  analysis,  are  found  to  contain 


4  CHEMICAL   BASI8   OF  THE   ANIMAL  BODY. 

some  reprevsentatives  of  carbohydrates  and  fats  as  well  as  of  pro- 
teids.  We  might  perhaps  even  go  as  far  as  to  say  that,  in  all 
forms  of  living  substance,  the  proteid  basis  is  found  upon  analysis 
to  have  some  carbohydrate  and  some  kind  of  fat  associated  with 
it.  Further,  not  only  does  the  normal  food  which  is.  eventually 
built  up  into  living  substance  consist  of  all  three  classes,  but,  as 
we  have  seen  in  the  sections  on  nutrition,  gives  rise  by  meta- 
bolism to  members  of  the  same  three  classes ;  and  as  far  as  we 
know  at  present,  carbohydrates  and  fats,  when  formed  in  the 
body  out  of  proteid  food,  are  so  formed  by  the  agency  of  living 
substance,  by  the  action  of  some  living  tissue.  Hence  there  is  at 
least  some  reason  for  thinking  it  probable  that  the  molecule  of 
living  substance,  if  we  may  use  such  a  phrase,  is  far  more  com- 
plex than  a  molecule  of  proteid  matter,  that  it  contains  in  itself 
residues  so  to  speak  not  only  of  proteid,  but  also  of  carbohydrate 
and  fatty  material. 

The  Plasmodium  of  ^tlialium  septicum,  a  myxomycaetous  fungus, 
presents  a  convenient  source  of  extremely  primitive  protoplasm  which 
may  be  obtained  in  large  quaaitities.  It  occurs  as  an  extended,  yel- 
low, gelatinous  mass,  frequently  of  considerable  thickness,  on  the 
surface  of  heaps  of  spent  tan  or  other  similar  decaying  vegetable 
matter,  and  exhibits  very  active  movements  both  internally  and  more 
particularly  at  its  edges,  of  an  essentially  amoeboid  nature.  It  has 
been  carefully  analysed  by  Reinke,  Studien  ilber  das  Protoplasma, 
Berlin,  1881.  See  also  Krukenberg,  Unters.  a.  d.  physiol.  Inst. 
Beidelb.,  Bd.  ii.  1882,  S.  273. 

Whether  this  be  so  or  not,  for  at  present  no  dogmatic  state- 
ment can  be  made,  there  is  no  doubt  that  when  we  examine  the 
various  tissues  and  fluids  of  the  animal  body  from  a  chemical 
point  of  view  we  find  present  in  different  places,  or  at  different 
times  in  the  same  tissue  or  fluid,  several  varieties  and  derivatives 
of  the  three  chief  classes ;  we  find  many  forms  of  proteids,  and 
bodies  closely  allied  to  proteids,  in  the  forms  of  mucin,  gelatine, 
&c. ;  many  varieties  of  fats  ;  and  several  kinds  of  carbohydrates. 

We  find,  moreover,  many  other  substances  which  we  may  re- 
gard as  stages  in  the  constructive  or  destructive  metabolism  of 
the  various  forms  and  phases  of  living  matter,  and  which  are  im- 
portant not  so  much  from  the  quantity  in  which  they  occur  in  the 
animal  body  at  any  one  time  as  from  their  throwing  light  on  the 
nature  of  animal  metabolism ;  these  are  such  substances  as  urea, 
uric  acid,  other  organic  crystalline  bodies,  and  the  extractives  in 
general. 

In  the  following  pages  the  chemical  features  of  the  more 
important  of  these  various  substances  which  are  known  to  occur 
in  the  animal  body  will  be  briefly  considered,  such  characters 
only  being  described  as  possess  or  promise  to  possess  physio- 


CHEMICAL   BASIS   OF   THE  ANIMAL   BODY.  5 

logical  interest.  The  physiological  function  of  any  substance 
must  depend  ultimately  on  its  molecular  (including  its  chemical) 
nature  ;  and  though  at  present  our  chemical  knowledge  of  the 
constituents  of  an  animal  body  gives  us  but  little  insight  into 
their  physiological  properties,  it  cannot  be  doubted  that  such 
chemical  information  as  is  attainable .  is  a  necessary  preliminary 
to  all  physiological  study. 

PKOTEIDS.i 

These  form  the  principal  solids  of  the  muscular,  nervous,  and 
glandular  tissues,  of  the  serum  of  blood,  of  serous  fluids,  and  of 
lymph.  In  a  healthy  cojidition,  sweat,  tears,  bile,  and  urine  con- 
tain mere  traces,  if  any,  of  proteids.  Their  general  percentage 
composition  may  be  taken  as  lying  within  the  following  limits:  — 

C  50-0  to  55-0 50-0  to  55-0 

H    6-9   „     7-3 6-8  „     7-3 

N  15-0  „  lS-0 15-4  „  18-2 

0  20-0  „  23-5 22-8  „  24-1 

S     0-3   „     2-0 0-4  „     5-0 

(Hoppe-Seyler.2)  (Drechsel.) 

The  composition  of  the  true  proteids  lies  so  constantly  within 
the  above  limits  that  conclusions  as  to  the  proteid  nature  of  any 
substance  whose  purity  is  assured  may  be  drawn  with  safety  from 
the  results  of  its  ultimate  analysis.  This  is  important  in  cases 
where  a  substance  is  with  difficulty,  if  at  all,  obtained  in  a  con- 
dition such  that  it  yields  none  of  the  reactions  characteristic  of 
proteids.  Klihne  and  Chittenden's  analyses  ^  of  peptones  freed 
from  albumoses,  which  they  quote  with  considerable  reserve, 
alone  show  a  percentage  composition  lying  appreciably  outside 
the  above  limits. 

In  addition  to  the  above  constituents,  proteids  ordinarily  leave  on 
ignition  a  variable  quantity  of  ash.  In  the  case  of  egg-albumin  the 
principal  constituents  of  the  ash  are  chlorides  of  sodium  and  potas- 
sium, the  latter  exceeding  the  former  in  amount.  The  remainder 
consists  of  sodium  and  potassium,  in  combination  with  phosphoric, 
sulphuric,  and  carbonic  acids,  and  very  small  qixantities  of  calcium, 
magnesium,  and  iron,  in  union  with  the  same  acids.  There  may  be 
also  a  trace  of  silica.'*     The  ash  of  serum-albumin  contains  an  excess 

^  The  chemistry  of  proteids  and  allied  substances,  together  with  a  compendious 
literature  of  the  subject,  is  very  fully  treated  and  recorded  in  Diechsel's  article 
"  Eiweisskorper  "  in  Ladenburg's  Handworferhnck  der  Cheniie,  Bd.  in.  (1885),  S.  534, 
and  in  Beilstein's  Handbuch  der  organischen  Chemie,  Bd.  iii.  (1882-90),  S.  1258. 

2  Hdbch.  d.  phys.  path.  chem.  Anal.  Auf.  5  (188.3),  S.  258. 

3  Zt.  f-  Biol.  Bd.  XXII.  (1886),  S.  452. 

4  Gmelin,  Hdbch.  d.  org.  Chem.  Bd.  viii.,  S.  285. 


6  PEOTEIDS. 

of  sodium  chloride,  but  the  ash  of  tlie  proteids  of  muscle  contains  an 
excess  of  potash  salts  and  phosphates.  The  nature  of  the  connection 
of  the  ash  with  the  proteid  is  still  a  matter  of  obscurity,  and  it  is  not 
known  whether  they  constitute  an  integral  part  of  its  molecule  or  are 
merely  adherent  impurities.  There  is  a  certain  amount  of  probability 
that  the  latter  is  the  case,  inasmuch  as  an  increasing  number  of  pro- 
teids  have  in  recent  times  been  obtained  practically  free  from  any 
ash-residue  on  ignition.  It  is,  however,  possible  that  in  their  natural 
condition  as  constituents  of  the  animal  tissues  and  fluids  the  proteids 
are  combined  with  salts,  the  separation  of  which  we  are  now  spealjiing 
being  an  artificial  result  of  the  i^rocesses  employed  to  effect  that 
separation.  The  sulphur  in  jjroteids  is  present  partly  in  a  stably 
combined  condition,  partly  loosely  combined.  The  latter  is  removed 
by  boiling  with  alkalis,  the  former  is  not.  The  proportions  of  the 
two  differ  in  the  several  proteids.^ 

Proteids  met  with  in  the  animal  body  are  all  amorphous,  the 
only  apparent  exception  being  haemoglobin  :  this  substance  is 
however  not  a  pure  proteid  but  a  compound  of  a  proteid  globin 
with  the  less  complex  haematin.  It  is  to  the  latter  that  the 
power  of  crystallising  is  due. 

Some  are  soluble,  some  insoluble  in  water,  some  are  character- 
istically soluble  in  moderately  concentrated  solutions  of  neutral 
salts,  and  all  are  for  the  most  part  insoluble  in  alcohol  and  ether ; 
they  are  all  soluble  in  strong  acids  and  alkalis,  but  in  becoming 
dissolved  mostly  undergo  decomposition.  Their  solutions  exert 
a  left-handed  rotatory  action  on  the  plane  of  polarisation,  the 
amount  depending  on  various  circumstances,  and  differing  for  the 
several  proteids. 

Crystals  into  whose  composition  certain  proteid  (globulin)  elements 
largely  entered  were  long  since  observed  in  the  aleurone-grains  of 
many  seeds.^  Similar  crystalloid  compounds  are  also  described  as 
occurring  occasionally  in  the  egg-yolk  of  some  animals  (Amphibia  and 
Fishes).  By  appropriate  methods  they  may  be  separated  and  re- 
crystallized  from  their  solution  in  distilled  water,  most  readily  by 
Drechsel's  method  of  alcohol  dialysis.^  The  crystals  consist  in  no 
case  of  pure  proteids,  but  are  always  compounds  of  the  latter  with 
some  inorganic  residue  such  as  lime  or  magnesia.  These  recrystal- 
lized,  and  hence  presumably  pure,  compounds  have  been  frequently 
analysed  with  a  view  to  establishing  a  formula  for  proteids  which 
should  give  some  clue  to  their  molecular  magnitude.  An  excellent 
summary  of  the  endeavours  to  arrive  at  a  definite  formula  for  proteids, 
based  on  the  above  analyses  and  on  those  of  haemoglobin  and  certain 
compounds  of  egg-albumin  with  salts  of  copper  and"  silver  is  given  by 

1  A.  Kriiger,  Pfliiger's  Arch.  Bd.  xliii.  (1888),  S.  244. 

-  For  literature  down  to  the  year  1877,  see  Weyl,  Zt.  f.  physlol.  C/i.  Bd.  i.,  S.  84. 
See  also  Hoppe-Seyler's  Handbiich,  Ed.  v.  p.  259.  Vines,  Jl.  of  Physiol.  Vol.  iii. 
(1880),  p.  102.     Chittenden  and  Hartwell, .//.  of  Physiol.  Vol.  xi.'  (1890),  p.  435. 

3  Jl.  f  prakt.  Chem.  N.  F.  Bd.  xix.  (1879).  S.  331. 


CHEMICAL   BASIS   OF  THE  ANIMAL   BODY.  7 

Bunge.^  As  the  result  of  these,  various  formulae  have  been  proposed 
by  the  several  observers.  Very  little  real  importance  can  however  be 
attached  to  these  formulae,  for,  as  Drechsel  observes,  in  so  large  a 
molecule  an  analytical  error  of  '01  p.c.  would  have  the  same  impor- 
tance as  would  one  of  -1  p.c.  iii  ordinary  analyses.  They  give  us  at 
most  an  idea  of  the  minimal  magnitude  of  the  proteid  molecule,  but 
apart  from  this  they  throw  no  more  light  on  the  subject  than  already 
existed  in  Lieberkuhn's  older  formula. 

General  reactions  of  the  proteids.'^ 

1.  Heated  with  strong  nitric  acid,  they  or  their  solutions  turn 
yellow,  and  this  colour  is,  on  the  addition  of  ammonia,  or  caustic 
soda  or  potash,  changed  to  a  deep  orange  hue.  (Xanthoproteic 
reaction.) 

If  much  proteid,  except  albumoses  and  peptones,  be  present  a 
yellow  precipitate  is  obtained  at  the  same  time.  With  less  pro- 
teid their  solutions  merely  turn  yellow^  on  boiling  and  orange  on 
the  addition  of  the  alkali :  if  only  a  trace  of  proteid  is  present  no 
yellow  colour  is  observed  until  after  the  addition  of  the  alkali. 

2.  With  Millon's  reagent  ^  they  give,  when  present  in  suffi- 
cient quantity,  a  precipitate,  which  turns  red  on  heating.  If  they 
are  only  present  in  traces,  no  precipitate  is  obtained,  but  merely 
a  red  colouration  of  the  solution  when  heated. 

3.  If  mixed  with  an  excess  of  concentrated  solution  of  sodium 
hydrate,  and  one  or  two  drops  of  a  dilute  solution  of  cupric  sul- 
phate, a  violet  colour  is  obtained,  which  deepens  in  tint  on  boil- 
ing.    (Piotrowski's  reaction.^) 

The  above  serve  to  detect  the  smallest  traces  of  all  proteids. 

4.  Eender  the  fluid  strongly  acid  with  acetic  acid,  and  add  a 
few-  drops  of  a  solution  of  ferrocyanide  of  potassium ;  a  precipi- 
tate shews  the  presence  of  proteids,  except  true  peptones  and 
some  forms  of  albumose. 

5.  Eender  the  fluid,  as  before,  strongly  acid  with  acetic  acid, 
add  an  equal  volume  of  a  concentrated  solution  of  sodium  sul- 
phate, and  boil.  A  precipitate  is  formed  if  proteids,  except  pep- 
tones, are  present. 

1  Lehrh.  d.  physiol.  u.  path.  Chem.  1887,  Su.  52-58.  For  most  recent  analysis  of 
haemoglobin  from  dog's  blood  see  Jaquet,  Zt.f.  physiol.  Ch.  Bd.  xii.  (1888),  S.  285, 
XIV.  S.  289.  Chittenden  and  Whitehouse,  Stud.  Lab.  physiol.  Chem.  Yale,  Vol.  ii. 
(1887),  p.  95. 

"^  Consult  in  all  cases  Hoppe-Seyler's  Hdbch  d.  physiol.  path.  chem.  Analyse,  iud. 
V.  1883.     See  also  Krukenberg,  Sitkb.  d.  Jena.  Gesell.f.  Med.  u.  Natwtss.  1885,  Nr.  2. 

3  Compt.  Rend.  T.  xxviii.  (1849),  p.  40. 

*  SItzb.  d.  Wien.  Akad.  Bd.  xxiv.  (1857),  S.  335. 


8  PEOTEIDS. 

This  reaction  is  particularly  useful,  not  merely  because  it  effects 
a  very  complete  precipitation  of  the  proteids  which  are  present  (except 
peptones),  but  also  because  the  reagents  employed  do  not  produce  any 
decomposition  of  other  substances  which  may  be  present,  and  do  not 
interfere  with  certain  other  tests  which  it  may  be  necessary  to  apply 
after  the  removal  of  the  proteids  by  filtration.  It  is  of  use  more  par- 
ticularly in  the  determination  of  sugar  in  blood. ^ 

The  following  reactions  are  specially  used  for  freeing  solutions 
from  all  proteids  by  precipitation. 

6.  Acidulate  faintly  with  acetic  acid  and  add  tannic  acid. 

7.  Acidulate  with  hydrochloric  acid  and  add  the  double  iodide 
of  mercury  and  potassium.     (Briicke's  reagent.^) 

8.  Add  hydrochloric  acid  until  the  reaction  is  strongly  acid ; 
then  add  phosphotungstic  acid. 

The  following  methods  are  often  additionally  useful  for  freeing 
solutions  from  all  proteids. 

i.  Precipitate  by  excess  of  absolute  alcohol,  having  previously 
made  the  solution  neutral  or  faintly  acid. 

ii.  Prepare  a  solution  of  ferric  acetate  by  saturating  acetic  acid 
with  freshly  precipitated  ferric  oxide,  avoiding  all  excess  of  free  acid. 
Add  this  to  the  solution  and  boil ;  the  whole  of  the  proteids  are  pre- 
cipitated together  with  the  iron;  the  latter  as  a  basic  salt.^  In  some 
cases  a  mixture  of  ferric  chloride  and  an  excess  of  sodium  acetate  is 
employed.^ 

iii.  Boil  the  solution  for  a  few  minutes  with  a  little  hydrated 
oxide  of  lead  in  presence  of  a  little  lead  acetate.^ 

In  recent  years  various  neutral  salts,  more  particularly  neutral 
ammonium  sulphate,^  have  been  largely  employed  for  effecting 
the  precipitation  and  separation  of  the  several  proteids. 

All  proteids  yield  a  characteristic  violet  colouration  with  simul- 
taneous slight  fluorescence  upon  treatment  with  glacial  acetic 
acid  and  strong  sulphuric  acid  (Adamkiewicz'  reaction).  The 
reaction  is  best  obtained  by  adding  to  the  suspected  solution  or 
substance  a  mixture  of  one  volume  of  strong  sulphuric  acid  and 
two  volumes  of  glacial  acetic  acid  and  boiling.'^     The  violet-col- 

1  See  Gamgee's  Physiol.  Chem.  Vol.  i.  p.  195. 

2  Sitzb.  d.  Wien.  Akad.  lxiii.  2  (1871),  Feb.  Hft. 

3  Hoppe-Seyler,  Hdbch.  S.  264. 

*  Seegen,  Pfliiger's  Arch.  Bd.  xxxiv.  (1884),  S.  391. 

^  Hofmeister,  "Zt.f.  physiol.  Chem.  Bd.  ii.  (1878),  S.  288. 

6  Wenz,  Zt.f.  Biol  Bd.  xxii.  (1886),  S.  10.  Kiihne,  Verkand.  d.  Naturhist.-Med. 
Ver.  Heidelb.  N.  F.  Bd.  in.  1885,  S.  286.  See  also  Halliburton,  Jl.  of  Physiol.  Vol.  v. 
(1883),  p.  172. 

■J  Hammarsten,  Pfliiger's  Arch.  Bd,  xxxvi.  (1885),  S.  389. 


CHEMICAL   BASIS   OF   THE   ANIMAL  BODY.  9 

oured  solution  observed  if  proteids  are  present  gives  an  absorption 
band  between  the  lines  &  and  F  in  the  solar  spectrum. 

No  general  method  can  be  given  for  the  quantitative  estimation 
of  the  various  proteids.  For  this  some  special  manuals  should  be 
consulted  and  use  made  of  the  reactions  which  are  specifically 
characteristic  of  each  proteid  as  given  below. 

Solutions  of  different  proteids  rise  to  different  heights  in  capillary 
tubes.  It  is  possible  that  this  fact  may  be  of  use  in  detecting  and 
estimating  their  approximate  relative  amounts.-^ 


Classification  of  the  Peoteids.^ 
The  following  classification  is  both  convenient  and  concise. 

Class  I.     Native  albumins. 

Soluble  in  distilled  water.  Solutions  coagulated  on  heating, 
especially  in  presence  of  a  dilute  (acetic)  acid.  Not  precipitated 
by  carbonates  of  the  alkalis  or  by  sodium  chloride,  or  generally 
by  solutions  of  neutral  salts. 

1.     Egg-albumin.     Serum-albumins. 

Class  II.     Derived  albumins  {Albuminates). 

Insoluble  in  distilled  water  and  in  dilute  neutral  saline  solu- 
tions ;  soluble  in  acids  and  alkalis.  Solutions  not  coagulated  by 
boiling- 

1.  Acid-albumin.  2.  Syntonin.  3.  Alkali-albumin.  4  Casein 
or  Native  alkali-albumin.^ 

Class  III.       Globulins. 

Insoluble  in  distilled  water,  soluble  in  dilute  saline  solutions. 
Soluble  in  very  dilute  acids  and  alkalis :  if  the  acids  and  alkalis 
are  strong  they  are  rapidly  changed  into  members  of  Class  II. 
Eeadily  precipitated  by  saturating  their  dilute  saline  solu- 
tions with  neutral  salts  such  as  sodium  chloride  or  magnesium 
sulphate. 

1  Bodlander  u.  Traube,  Ber.  d.  deutsch.  chem.  Gesell  Bd.  xix.  (1886),  S.  1871. 

•^  See  Hoppe-Seyler,  Hdhcli  Ed.  v.  S.  265.  Drechsel  in  Ladenburg's  Handworter- 
buck  d.  Chem.  Bd.  iii.  S.  550.  Danilewski,  ^rc^.  d.  Sci.  phjs.  et  nat.  {3)  T.  7  (1882), 
Nr.  4. 

^  Casein  differs  in  many  respects  from  the  other  members  of  this  class,  but  in  its 
general  reactions  is  more  closely  allied  to  them  than  to  the  members  of  any  other 
class.  In  its  ready  precipitability  by  neutral  salts  it  shews  some  affinity  to  the 
globulins. 


10  PROTEIDS. 

1.  Crystallin,  the  globulin  of  the  crystalline  lens.  2.  Vitellin. 
3.  Paraglobulin  or  Serum-globulin.  4.  Fibrinogen.  5.  Myosin. 
6.    Globin. 

Class  IV.     Fibrins. 

Insoluble  in  water.  Soluble  with  difficulty  in  strong  acids  and 
alkalis,  and  undergoing  a  simultaneous  change  into  members  of 
Class  II.  Soluble  by  the  prolonged  action  of  moderately  strong 
(10  p.c.)  solutions  of  neutral  salts,  with  simultaneous  change  into 
members  of  Class  III. 


Class  V.      Coagulated  proteids. 

Products  of  the  action  of  heat  on  members  of  Classes  I.,  III., 
and  IV.,  or  of  Class  II.  when  precipitated  by  neutralisation  and 
heated  in  suspension  in  water.  They  are  also  obtained  by  the 
prolonged  action  of  alcohol  in  excess  upon  members  of  Classes  I., 
III.,  and  IV.  Their  solubilities,  except  in  solutions  of  neutral 
salts,  are  in  general  similar  to,  but  less  than  those  of  Class  IV. 


Class  VI.     Albunioses  and  pejJtones.^ 

The  true  peptones  are  extremely  soluble  in  water.  They  are 
not  precipitated  by  acids,  alkalis,  neutral  salts,  or  many  of  the 
reagents  which  precipitate  other  proteids.  They  are  precipitated 
but  not  coagulated  by  even  the  prolonged  action  of  alcohol.  Pep- 
tones are  readily  diffusible,  albumoses  less  so.  Some  of  the  albu- 
nioses are  readily  soluble  in  water,  some  are  less  soluble.  They 
are  distinguished  from  peptones  by  being  precipitated  when  their 
solutions  are  saturated  with  neutral  ammonium  sulphate.  They 
yield  precipitates  with  many  of  the  reagents  which  precipitate 
other  proteids,  and  it  is  specially  characteristic  that  the  precipi- 
tates they  yield  with  nitric  acid  and  with  ferrocyanide  of  po- 
tassium in  presence  of  acetic  acid  disappear  when  warmed  and 
reappear  on  cooling. 


Class  VII.     Lardacein  or  amyloid  sxibstance. 

Insoluble  in  water,  dilute  acids  and  alkalis,  and  saline  solu- 
tions. Converted  into  members  of  Class  II  by  strong  acids 
and  alkalis. 

1  The  albumoses  are  classed  with  the  peptones  partly  from  their  close  relationship 
to  these  substances  and  partly  for  convenience. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  11 

The  Chemistky  of  the  several  Proteids.^ 

Class  I.     Native  Albumins. 

1.     Egg-albumin. 

As  obtained  in  the  solid  form  by  evaporating  its  solutions  to 
dryness  at  40°,  preferably  in  vacuo,  it  forms  a  semi-transparent, 
brittle  mass,  of  a  pale  yellow  colour,  tasteless  and  inodorous.  Dis- 
solved in  water  it  yields  a  clear  neutral  colourless  solution.  This 
solution  coagulates  on  heating,  but  the  temperature  at  which  the 
coagulation  takes  place  varies  considerably  with  the  concentration, 
and  is  largely  dependent  upon  the  presence  or  absence  of  salts. 
The  more  commonly  observed  temperature  is  70-73°,  but  Gautier 
states^  that  coagula  may  also  be  obtained  at  54°  and  63°.  The 
more  dilute  the  solution  is,  the  higher  is  the  temperature  at  which 
it  coagulates,  thus  finally  resembling  a  solution  of  albumin  from 
which  the  salts  have  been  removed  by  dialysis.^  When  pre- 
cipitated from  solution  by  excess  of  alcohol  it  is  readily  coagu- 
lated by  the  precipitant,  so  that  it  is  now  usually  insoluble  in 
water.  In  this  respect  it  differs  somewhat  characteristically  from 
serum-albumin,  which  is  not  so  immediately,  though  it  is  ulti- 
mately, coagulated  by  the  action  of  alcohol. 

According  to  Coriu  and  Berard,*  by  applying  the  method  of  frac- 
tional heat-coagulation  to  filtered  white  of  egg,  coagula  may  be  ob- 
tained at  57-5°,  67°,  72°,  76°,  and  82°,  the  first  two  being  due  to 
globulins,  the  others  to  albumins. 

Strong  acids,  especially  nitric  acid,  cause  a  coagulation  similar 
to  that  produced  by  heat  or  by  the  prolonged  action  of  alcohol ; 
the  albumin  becomes  profoundly  changed  by  the  action  of  the 
acid,  and  does  not  dissolve  upon  removal  of  the  acid.  Mer- 
curic chloride,  nitrate  of  silver,  and  lead  acetate,  precipitate  the 
albumin,  forming  with  it  insoluble  compounds  of  variable  com- 
position. 

Strong  acetic  acid  in  excess  gives  no  precipitate,  but  when  the 
solution  is  concentrated  the  albumin  is  transformed  into  a  trans- 
parent jelly.  A  similar  jelly  is  produced  when  strong  caustic 
potash  is  added  to  a  concentrated  solution  of  egg-albumin.     In 

1  In  addition  to  the  works  already  quoted  consult  Beilstein,  Hdhch.  d.  org.  Chem. 
Bd.  III.  (1889),  Sn.  1258-1310,  for  all  data  concerning  the  proteids. 

^  Chimie  appliquee  a  la  Physiol,  ^-c.  T.  i.  (1874),  p.  242.  Haycraft  and  Duggan, 
Proc.  Roy.  Soc.  Edinb.  1889,  p.  364.  Starke  (Swedish).  See  Abst  in  Maly's  Jalires- 
bericht,  xi.  (1881),  S.  19. 

^  Laptschinsky,  Sitzh.  d.  Wien.  Akad.  Bd.  lxxvi.  1877.     Juli-Hfl. 

*   Travaux  du  Lab.  de  Leon  Fre'de'ricq,  Liege.     T.  ii.  (1888),  p.  170. 


12  PROTEIDS. 

both  these  cases  the  substance  is  profoundly  altered,  becoming  in 
the  one  case  acid-  in  the  other  alkali-albumin. 

The  specific  rotatory  power,  which  is  stated  to  be  independent 
of  the  concentration,  is  variously  given  as  (a)D=  — 35-5°  (Hoppe- 
Seyler),  or  —  37 '79°  (Starke).  The  latter  agrees  closely  with  Haas' 
determination^  (a)D  =  — 38-1°  and  is  probably  the  most  correct  of 
the  three  values. 

Preparations.  The  fibrous  network  in  white  of  egg  is  broken 
up  with  scissors  and  violently  agitated  in  a  flask  till  a  thick  froth 
is  formed.  The  flask  is  then  inverted,  whereupon  the  foam  rises 
to  the  top,  carrying  the  larger  part  of  the  fibrous  debris  with  it. 
The  clear  subnatant  fluid  is  now  carefully  drawn  off  and  filtered 
through  fine  muslin ;  to  this  an  equal  volume  of  water  is  added, 
and  the  whole  is  finally  filtered  through  coarse  paper.  From  this 
point  onwards  two  methods  may  be  employed. 

1.  For  ordinary  purposes  the  fluid  may  be  very  carefully  and 
faintly  acidulated  with  acetic  acid,  filtered  and  the  filtrate  purified 
by  dialysis. 

2.  To  obtain  the  purest  albumin  proceed  as  follows :  ^  Saturate 
the  fluid  with  magnesium  sulphate  at  20°,  filter  and  saturate  the 
filtrate  with  sodium  sulphate.  Dissolve  the  precipitate  of  albu- 
min thus  obtained  in  water  and  precipitate  again  with  the  sodium 
salt,  and  after  repeating  this  process  several  times  remove  the 
last  traces  of  salt  by  dialysis  and  concentrate  to  dryness  at  40°. 

According  to  recent  researches  egg-albumin  may  be  obtained  in 
a  crystalline  form  by  slow  evaporation  of  its  solutions  in  presence 
of  neutral  ammonium  sulphate.  The  separation  takes  place  at 
first  in  the  form  of  minute  spheroidal  globules  of  various  sizes,  and 
finally  minute  needles,  either  aggregated  or  separate,  make  their 
appearance.  It  has  not  as  yet  been  found  possible  to  obtain 
these  so-called  crystals  from  solutions  which  have  been  freed 
by  dialysis  from  the  ammonium  salt.  Further  investigation  is 
needed  to  establish  their  real  nature.^ 

The  primary  digestive  products  obtained  during  the  peptic  diges- 
tion of  egg-albumin  have  been  studied  b}"  Chittenden  and  Bolton.'* 

2.     Serum-albumin. 

This  is  the  sole  proteid,  apart  from  the  globulins,  which  occurs 
in  serum.^     Pure  solutions  of  this  proteid  closely  resemble  those 

^  Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  378. 

2  Starke,  loc.  cit. 

3  Hofmeister,  Zt.f.  phjsiol.  Chem.  Bd.  xiv.  (1889)  S.  16.5.  Gabriel,  ibid.  Bd.  xv. 
Hf.  5  (1891)  S.  456. 

*  Stud.  Lah.  Physiol.  Chem.  Yak  Univ.  Vol.  ii.  (1887),  p.  126. 

^  '  Serum  casein  '  of  Kiihne  and  Eichwald  was  shewn  by  Hammarsten  to  consist 


CHEMICAL  BASIS   OF  THE   AJTIMAL  BODY.  13 

of  egg-albumin  in  their  general  reactions,  but  the  difference  of 
the  two  is  clearly  shewn  by  the  following  statements :  — 

1.  When  free  from  salts  and  in  1 — 1-5  p.c.  solution  it  coaou- 
lates  on  heating  to  50°.  The  addition  of  sodium  chloride  raises 
the  coagulating  point  to  75° — 80°. ^  Under  the  conditions  in 
which  it  occurs  in  serum  it  is  not  found  to  shew  any  opalescence 
on  heating  at  any  temperature  below  60°,  and  it  may  be  regarded 
as  coagulating  completely  at  75.° 

By  fractional  heat-coagulation  of  serum  freed  from  globulin  Halli- 
burton ^  has  obtained  evidence  of  the  existence  in  the  serum  of  many 
animals  of  three  albumins  coagulating  at  70-73°,  77-78°,  and  82-85°. 
In  some  serum  only  two  of  these  albumins  occur. 

2.  It  is  not  readily  coagulated  by  alcohol  or  precipitated  by 
ether :  egg-albumin  is,  and  most  readily  by  alcohol. 

3.  It  is  difficult  to  make  any  one  definite  statement  as  to  the 
specific  rotatory  power  of  serum-albumin  since  it  appears  to  differ 
for  the  substance  as  obtained  from  different  animals.  Starke 
gives  it  as  (a)D  =  — 62-6°  for  human  serum-albumin,  and  —60-05° 
for  that  of  the  horse. 

4.  It  is  not  very  readily  precipitated  by  strong  hydrochloric 
acid,  and  the  precipitate  is  readily  soluble  on  the  further  addition 
of  acid  :  the  reverse  is  the  case  for  egg-albumin. 

5.  Precipitated  or  coagulated  serum-albumin  is  more  readily 
soluble  in  nitric  acid  than  is  eo-or-albumin. 


■"0& 


6.  When  precipitated  by  alcohol  it  is,  as  already  stated,  less 
immediately  though  it  is  ultimately  coagulated  by  the  action  of 
the  precipitant,  than  is  egg-albumin. 

7.  According  to  Gauthier^  the  following  reagent  precipitates 
egg-albumin  but  not  serum-albumin :  250  c.c.  caustic  soda,  sp.  gr. 
0-7 :  50  c.c.  sulphate  of  copper  1  p.c. :  700  c.c.  glacial  acetic  acid. 
To  be  added  in  the  ratio  of  10  c.c.  to  2  c.c.  of  the  fluid  to  be 
tested. 

8.  Egg-albumin  if  injected  subcutaneously  or  into  a  vein,  re- 
appears unaltered  in  the  urine ;  serum-albumin  similarly  injected 
does  not  thus  normally  pass  out  by  the  kidney. 

Seruni-albumin  is  found  not  only  in  blood-serum,  but  also  in 
lymph,  both  that  contained  in  the  proper  lymphatic  channels  and 

really  of  serum-globulin,  and  this  is  confirmed  by  Halliburton,  Jl.  Physiol.  Vol.  v. 
(1883),  p.  193. 

1  Starke,  loc.  cit.  S.  18. 

2  Jl.  Physiol.  Vol.  v.  1883,  p.  152.     But  see  also  Vol.  xi.  (1890),  p  453. 
*  See  M'alv's  Ber.  Bd.  xv.  (1885),  S.  31. 


14  PROTEIDS. 

that  diffused  in  the  tissues ;    in  chyle,  milk,  transudations,  and 
many  pathological  fluids. 

It  is  this  form  in  which  albumin  generally  appears  in  the  urine. 

Scherer  described  two  proteids  which  he  obtained  from  the  contents 
of  ovarial  cysts,  and  to  which  he  gave  the  names  of  metalbumin  and 
paralbumin.-'  Hammarsten  concludes  from  his  researches  ^  that  they 
are  really  identical.  Metalbumin  seems  to  be  associated  with  some 
carbohydrate  substance  resembling  glycogen  (?),  since  it  yields,  on 
heating  with  sulphuric  acid,  a  body  which  reduces  Fehling's  fluid  as 
does  dextrose.^ 

Neither  egg-  nor  serum-albumin  can  be  obtained  in  a  condition  such 
that  they  leave  no  ash  residue  on  ignition.  Al.  Schmidt  asserted^ 
that  they  could  be  by  means  of  dialysis,  and  that  in  this  condition 
they  were  no  longer  coagulable  by  lieat.  On  this  point  a  keen  con- 
troversy vv^as  carried  on  for  some  time,  for  the  details  of  which  see 
Eollett's  article  on  Blood  in  Hermann's  Hdbch.  d.  Physiol.  Bd.  iv. 
Th.  1,  S.  93.  The  whole  difliculty  seems  to  have  turned  on  the  ex- 
treme sensitiveness  of  dialysed  solutions  of  albumin  to  the  presence 
or  absence  of  traces  of  acid  or  alkali,  and  on  the  fact  that  such  dialysed 
albumin  is  largely  changed  into  an  albuminate.^ 

Preparations  of  pure  serum-albumin.  Centrifugalised  serum  is 
saturated  at  30°  with  magnesium  sulphate,  and  the  precipitated 
globulin  ^  is  washed  on  the  filter  with  a  saturated  solution  of  the 
salt.  The  filtrates  are  then  saturated  at  40°  with  sodium  sul- 
phate; by  this  means  the  serum-albumin  is  precipitated.  The 
precipitate  is  dissolved  in  water,  reprecipitated  by  sodium  sul- 
phate, and  the  process  repeated  several  times.  The  final  product 
is  then  freed  from  salts  by  dialysis,  precipitated  by  excess  of 
alcohol,  washed  with  this,  and  finally  with  ether,  and  dried  by 
exposure  to  the  air.'' 

The  facts  on  which  this  method  is  based  were  clearly  stated  by 
Denis.®  Schafer  rediscovered^  the  precipitability  of  serum-albumin 
by  sodium  sulphate  in  presence  of  the  magnesium  salt.  Halliburton 
has  shewn  ^°  that  this  is  due  to  the  action  of  the  double  sulphate  of 
magnesium  and  sodium  MgNag (804)2  6  HgO. 

1  Ann.  d.  Chem.  u.  Pharm.  Bd.  82  (1852),  S.  135. 

2  Maly's  Ber.  Bd.  xi.  (1881),  S.  11.     Zt.  physio!.  Ch.  Bd.  vi.  (1882),  S.  194. 

3  Landwehr,  Pfliiger's  Arch.  Bd.  xxxix.  (1886),  S.  203.  Zt.  phijsioL  Chem.  Bd. 
VIII.  (1883),  S.  114.  Hilger,  AnnaL  d.  Chem.  Bd.  160  (1871),  S.  338.  Pldsz,  Hoppe- 
Seyler's  Med.-Chem.  Unters.  (1871),  S.  517.  Obolensky,  Pfliiger's  Arch.  Bd.  iv. 
(1871),  S.  346. 

4  Pfliiger's  Arch,  xi,  (1875),  S.  1. 

5  Werigo,  Pfliiger's  Arch,  Bd.  xlviii.  (1890),  S.  127. 

6  Hammarsten,  Zt.  f.  vhijsiol.  Ch.  Bd.  viii.  (1884),  S.  467. 
■^  Starke,  loc.  cit.  (sub.  egg-albumin),  S.  18. 

8  Etudes  sur  le  sang,  Paris,  1859,  p.  39. 

9  .//.  of  Physiol.  Vol.  III.  (1880),  p.  184. 
10  Ibid.  Vol.  V.  1883,  p.  181. 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.  15 

Class  II.     Derived  Albumins  {Albuminates). 

1.    Acid-albumin. 

When  a  native  albumin  in  solution,  such  as  egg-  or  serum- 
albumin,  is  treated  for  some  little  time  with  a  dilute  acid,  such 
as  hydrochloric,  its  properties  become  entirely  changed.  The 
most  marked  changes  are  (1)  that  the  solution  is  no  longer  coag- 
ulated by  heat;  (2)  that  when  the  solution  is  carefully  neutral- 
ised the  whole  of  the  proteid  is  thrown  down  as  a  precipitate ;  in 
other  words,  the  serum-albumin,  which  was  soluble  in  water,  or 
at  least  in  a  neutral  fluid  containing  only  a  small  quantity  of 
neutral  salts,  has  become  converted  into  a  substance  insoluble  in 
water  or  in  similar  neutral  fluids.  The  body  into  which  serum- 
albumin  thus  becomes  converted  by  the  action  of  an  acid  is 
spoken  of  as  acid-albumin.  Its  characteristic  features  are  that  it 
is  insoluble  in  distilled  water,  and  in  neutral  saline  solutions, 
such  as  those  of  sodic  chloride,  that  it  is  readily  soluble  in  dilute 
acids  or  dilute  alkalis,  and  that  its  solutions  in  acids  or  alkalis 
are  not  coagulated  by  boiling.  When  suspended,  in  the  undis- 
solved state,  in  water,  and  heated  to  75°  C,  it  becomes  coagulated, 
and  is  then  undistinguishable  from  coagulated  serum-albumin,  or 
indeed  from  any  other  form  of  coagulated  proteid.  It  is  evident 
that  the  substance  when  in  solution  in  a  dilute  acid  is  in  a  dif- 
ferent condition  from  that  in  which  it  is  when  precipitated  by 
neutralisation.  If  a  quantity  of  serum-  or  egg-albumin  be  treated 
with  dilute  hydrochloric  acid,  it  will  be  found  that  the  conversion 
of  the  native  albumin  into  acid-albumin  is  gradual ;  a  specimen 
heated  to  .75°  C.  immediately  after  the  addition  of  the  dilute  acid, 
will  coagulate  almost  as  usual ;  and  another  specimen  taken  at 
the  same  time  will  give  hardly  any  precipitate  on  neutralisation. 
Some  time  later,  the  interval  depending  on  the  proportion  of  the 
acid  to  the  albumin,  on  temperature,  and  on  other  circumstances, 
the  coagulation  will  be  less,  and  the  neutralisation  precipitate 
will  be  considerable.  Still  later  the  coagulation  will  be  absent, 
and  the  whole  of  the  proteid  will  be  thrown  down  on  neutrali- 
sation. 

The  conversion  of  the  native  albumins  in  solution  into  acid-albumin 
by  dilute  acids  is  facilitated  by  heating  to  temperatures  below  those 
at  which  the  albumins  respectively  coagulate.^  The  conversion  is  ex- 
tremely rapid  if  a  strong  acid  is  added  to  a  concentrated  solution  of 
the  proteid;  thus  when  a  little  glacial  acetic  acid  is  stirred  into  undi- 
luted white  of  egg  the  whole  solidifies  into  a  yellow  transparent  jelly 

1  Rollett,  Sitzh.  d.  Wien.  Alcad.  Bd.  lxxxiv.  (1881),  S.  332.  Hevnsins,  Pfluger's 
Arch.  Bd.  xi.  (1875),  S.  624. 


16  FEOTEIDS. 

consisting  of  acid-albumin.  A  similar  jelly  is  formed,  only  gradually, 
if  the  albumin  is  placed  in  a  ring-dialyser  and  floated  on  dilute  acids 
(1-2  p.  c.)^ 

Globulins  are  more  readily  converted  into  acid-albumin  than 
are  the  native  albumins.  Coagulated  proteids  or  fibrin  require 
for  their  conversion  the  application  of  the  acids,  preferably  hydro- 
chloric, in  a  concentrated  form,  the  products  thus  obtained  being 
practically  indistinguishable  from  the  products  of  the  action  of 
dilute  acids  on  the  more  readily  convertible  proteids.  As  ob- 
tained by  the  action  of  acids  on  the  various  proteids  the  products 
exhibit  certain  not  very  marked  differences,  which  however  in- 
dicate that  each  proteid  yields  its  own  special  acid-albumin.  The 
researches  of  Morner  ^  have  shewn  that,  contrary  to  earlier  views,-^ 
acid-albumins  differ  distinctly  from  the  alkali-albumins.  These 
differences  may  be  more  appropriately  considered  after  the  prep- 
aration and  properties  of  the  latter  have  been  described. 

Prepctration  1.  Serum  or  diluted  white  of  egg  is  digested  at 
40 — 50°  for  several  hours  with  1 — 2  p.c.  hydrochloric  acid.  The 
solution  is  now  filtered,  carefully  neutralised,  the  precipitate  col- 
lected on  a  filter  and  washed  with  distilled  water. 

2.  Acid-albumin  may  be  rapidly  prepared  by  adding  glacial 
acetic  acid  to  white  of  egg  which  has  been  chopped  with  scissors 
and  strained  through  muslin.  A  jelly  is  thus  formed  which  can 
be  dissolved  in  warm  water,  and  from  this  solution  the  acid-albu- 
min can  be  precipitated  by  neutralisation  and  washed  as  before. 

2.     Syntonin. 

Although  this  substance  is  merely  the  acid-albumin  which  re- 
sults from  the  action  of  acids  on  the  globulin  (myosin)  contained 
in  muscles,  and  in  its  more  obvious  properties  is  at  first  sight 
identical  with  other  acid-albumins,  it  merits  a  short  and  separate 
description,  not  only  on  account  of  its  historical  interest  in  the 
chemistry  of  muscles,  but  also  because  recent  work  has  shewn  it 
to  be  distinctly  different  from  the  similar  products  of  the  action  of 
acids  on  other  proteids,  and  its  properties  and  reactions  have  been 
more  fully  studied  than  those  of  any  other  form  of  acid-albumin. 

Liebig,  unacquainted  with  the  existence  of  myosin  in  the  dead 
muscle,  was  the  first  to  prepare  it  by  the  action  of  dilute  (-1  p.c.)  hy- 
drochloric acid  on  the  miiscle  substance,*  and  he  regarded  it  as  the 

1  Johuson,  J^?.  Chem.  Soc.  1874,  p.  734.  Ber.  d.  deutsch.  chem.  Gesell.  1874,  S.  826. 
RoUett,  luc.  cit. 

2  The  original  is  in  Swedish,  but  is  fully  abstracted  in  Maly's  Jahresbericht,  Bd. 
VII.  (1877),  S.  9,  and  is  also  published  in  extenso  in  rfliiger's  Arch.  Bd.  xvii.  (1878), 
S.  468.    A  convenient  resume  is  given  on  p.  .541 . 

8  Soyka,  Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  347. 
*  Annalen  d.  Chem.  u.  Phnrm.  Bd.  1^  (1850),  S.  125. 


CHEMICAL  BASIS   OF   THE   AMIMAL   BODY.  17 

chief  and  characteristic  proteid  of  muscles  (muscle-fibrin).  Kuhne, 
however,  shewed  in  his  famous  researches  on  muscle-plasma^  that  its 
formation  is  due  to  the  conversive  action  of  the  acid  on  myosin. 

Preparation.  By  the  action  of  0"1  p.c.  hydrochloric  acid  on 
pure  myosin  (see  below),  or  by  treatment  of  finely  chopped  and 
thoroughly  washed  muscle  substance,  preferably  from  the  frog, 
with  the  same  acid.  It  may  be  precipitated  from  its  solution  by 
neutralisation,  and  freed  from  salts  by  washing,  but  in  this  case 
care  must  be  exercised  as  to  the  extent  of  the  washing,  since  syn- 
tonin  is  distinctly  altered  by  the  prolonged  action  of  water,  espe- 
cially as  regards  its  solubility  in  dilute  acid  and  lime-water.^ 

The  reactions  specially  characteristic  of  this  substance  and  its 
distinction  from  other  forms  of  acid-albumin  and  from  alkali- 
albumin  are  indicated  in  the  following  statements.^ 

1.  It  is  soluble  in  lime-water,  and  this  solution  is  coagulated, 
though  incompletely,  by  boiling  (Kiihne). 

2.  It  is  insoluble  in  acid  phosphate  of  soda  (NaH2P04)  ;  other 
acid-albumins  are  soluble  (Morner).  In  presence  of  this  salt  it 
does  not  pass  into  solution  on  the  addition  of  alkali  until  the 
whole  of  the  acid  phosphate  has  been  converted  into  the  neutral 
(Na2HP04).  In  this  respect  it  differs  from  alkali-albumin,  which 
is  soluble  under  the  same  conditions  long  before  the  conversion  of 
the  acid  into  the  neutral  phosphate  is  complete. 

3.  It  is  soluble  in  dilute  sodium  carbonate. 

4.  When  precipitated  from  its  acid  solution  by  neutralisation 
the  precipitate  is  more  gelatinous  than  that  of  the  other  acid- 
albumins,  and  less  readily  soluble  in  alkalis  (Morner). 

5.  Its  specific  rotatory  power  when  dissolved  in  dilute  hydro- 
chloric acid  or  sodium  carbonate  is  independent  of  the  concentra- 
tion, and  is  given  as  (a)^  =  —  72°  (Hoppe-Seyler). 

Syntonin  has  been  stated  to  be  capable  of  reconversion  into  myosin, 
or  some  globulin  closely  resembling  it,  by  solution  in  lime-water,  ad- 
dition of  ammonium  chloride  to  an  amount  just  short  of  saturation, 
and  neutralisation  with  acetic  acid.  The  neutral  fluid  thus  finally 
obtained  is  allowed  to  fall  drop  by  drop  into  distilled  water,  from 
which  a  fine  coagulum  gradually  separates  out  consisting  of  myosin.* 
Hoppe-Seyler  states  that  by  similar  treatment  all  forms  of  acid-albu- 
min may  be  converted  into  globulins  resembling  myosin.^ 

1   Ueber  das  Protoplasma,  Leipzig,  1864,  S.  15. 

^  Kiihne,  loc.  cit.  S.  16.     Sander,  Arch.f.  Physiol.  Jalirg.  1881,  S.  198. 
^  See  Morner,  loc.  cit. 

*  A.  Danilewsky,  Zt.f.  physiol.  Ckem.  Bd.  v.  (1881),  S.  158. 
5  Hdbch.  d.  ahem.  Anal.  Ed.  v.  (1883),  S.  281. 

2 


18  PKOTEIDS. 


3.     Alkali-albumin. 

If  serum-  or  egg-albumin  or  washed  muscle  be  treated  with 
(Jilute  alkali  instead  of  with  dilute  acid,  the  proteid  undergoes  a 
change  in  many  ways  similar  to  that  which  was  brought  about  by 
the  acid.  The  alkaline  solution,  when  the  change  has  become 
complete,  is  no  longer  coagulated  by  heat,  the  proteid  is  wholly 
precipitated  on  neutralisation,  and  the  precipitate,  insoluble  in 
water  and  in  neutral  solutions  of  sodium  chloride,  is  readily  solu- 
ble in  dilute  acids  or  alkalis. 

Alkali-albumin  may  be  prepared  by  the  action  not  only  of 
dilute  alkalis  but  also  of  strong  caustic  alkalis  on  native  albumins 
as  well  as  on  coagulated  albumin  and  other  proteids.  The  jelly 
produced  by  the  action  of  caustic  potash  on  white  of  egg  (p.  11) 
is  alkali-albumin ;  the  similar  jelly  produced  by  strong  acetic  acid 
is  acid-albumin. 

In  short,  the  general  statement  may  be  made  that  under  other- 
wise similar  conditions,  if  an  alkali  is  employed  instead  of  an 
acid  to  act  on  proteids,  alkali-albumin  is  formed  instead  of  acid- 
albumin.  In  the  opinion  of  many  authors  ^  the  precipitates  ob- 
tained by  neutralising  the  acid  or  alkaline  solutions  which  arise 
during  the  preparation  of  acid-  and  alkali-albumin  respectively 
are  to  be  regarded  as  identically  the  same.  According  to  this 
view  the  neutralisation  precipitate  is  itself  neither  acid-  nor  alkali- 
albumin,  but  becomes  either  the  one  or  the  other  by  solution  in 
either  an  acid  or  alkali,  entering  at  the  same  time  into  union  with 
the  acid  or  alkali. 

Danilewsky  ^  has  utilised  the  tropaeolins  for  the  purpose  of  deter- 
mining the  fixation  of  acids  or  alkalis  by  proteids,  and  on  this  he  has 
based  a  classification  of  these  substances.  The  tropaeolins  are  soluble 
in  water,  the  one  (tropaeolin  00)  yielding  a  yellow,  the  other  (tro- 
paeolin  000  No.  1)  an  orange  solution.  The  first  is  changed  to  a 
lilac  colour  by  acids,  but  not  by  salts  which  have  an  acid  reaction  to 
litmus.  The  second  is  turned  to  bright  carmine  by  free  alkalis,  but 
not  by  salts  which  have  an  alkaline  reaction  to  litmus. 

It  is  however  on  the  whole  more  probable^  that  acid-  and 
alkali-albumin  are  distinct,  though  very  closely  allied  substances, 
and  we  might  go  even  so  far  as  to  say  that  probably  every  proteid 
yields  its  own  kind  of  either  the  one  or  the  other  proteid  on  treat- 
ment with  acids  and  alkalis.  But  as  yet  we  do  not  possess  any 
means  of  distinguishing  between  the  several  forms  of  each  sub- 
stance by  any  ordinary  reactions. 

1  Soyka,  Pfluger's  Arch.  xii.  (1876),  S.  347. 

2  Centralh.f.  d.  med.  Wiss.  1880,  No.  51. 

3  Murner,  Pfluger's  Arcli.  Bd.  xvii.  (1878),  S.  468.  But  see  also  Kieseritzky, 
Inaug.-Diss.,  Dorpat,  1882.  Abstr.  in  Maly's  Jakresber.  Bd.  xii.  (1882),  S.  6,  and 
Eosenberg,  Inaug.-Diss.,  Dorpat,  1883.     Abstr.  in  ]Vlaly,  Bd.  xiii.  (1883),  S.  19. 


CHEMICAL   BASIS    OF   THE   ANIMAL   BODY.  19 

The  chief  though  somewhat  unsatisfactory  evidence  which  is 
advanced  as  to  the  difference  of  the  two  products  is  the  f ollowino- : 

1.  Alkali-albumin  is  in  general  more  soluble  than  acid-albumin. 

2.  When  precipitated  by  neutralisation  the  former  (alkali)  is 
flocculent,  the  latter  (acid)  is  more  viscid,  transparent,  and  ge- 
latinous. 

3.  When  dissolved  in  a  minimum  of  alkali  and  heated  to 
100°  in  sealed  tubes,  alkali-albumin  coagulates,  acid-albumin 
does  not. 

4.  When  alkali-albumin  is  dissolved  in  NagHPOi  it  is  not  pre- 
cipitated on  the  addition  of  an  acid  until  all  the  salt  has  been 
converted  into  NaHaPOi.i     (Cf.  above,  p.  17.) 

5.  Acid-albumin  can  be  converted  into  alkali-albumin  by  the 
action  of  strong  alkalis,  but  the  reverse  conversion  of  the  product 
thus  obtained  or  of  an  ordinarily  prepared  alkali-albumin  into 
acid-albumin  is  stated  to  be  impossible. 

The  rotatory  power  of  alkali-albumin  varies  according  to  its 
source ;  thus  when  prepared  by  strong  caustic  potash  from  serum- 
albumin,  the  rotation  rises  from  -  56°  (that  of  serum-albumin) 
to  -86°,  for  yellow  light.  Similarly  prepared  from  egg-albumin, 
it  rises  from  -  38"5°  to  -47°,  and  if  from  coagulated  white  of  egg, 
it  rises  to  -  58'8°.  Hence  the  existence  of  various  forms  of  alkali- 
albumin  is  probable. 

The  substance  'protein,'  described  by  Mulder,^  appears,  if  it 
exists  at  all,  to  be  closely  connected  with  this  body.  All  sub- 
sequent observers  have  however  failed  to  confirm  his  views, 
and  it  is  only  mentioned  here  from  its  historical  interest.  Since 
Mulder's  time  the  name  has  been  applied  to  various  forms  of 
proteid. 

Preparation.  The  best  method  is  that  originally  introduced  by 
Lieberkiihn.^  Purified  white  of  egg  (see  p.  11)  is  made  into  a 
jelly  by  the  addition  with  rapid  stirring  of  strong  caustic  soda, 
avoiding  as  far  as  possible  all  excess  of  the  latter.  The  jelly  is 
then  cut  into  small  lumps  and  washed  in  distilled  water,  fre- 
quently changed,  until  the  lumps  are  quite  white  throughout. 
The  lumps  of  purified  albumin  are  then  dissolved  in  water  by 
gently  heating  on  a  water-bath,  the  solution  filtered,  and  the 
alkali -albumin  precipitated  by  careful  addition  of  acetic  acid. 
The  precipitate  is  then  thoroughly  washed  with  distilled  water. 

1  Soyka,  he.  cit.     See  also  Soxhlet,  Jn.  f.  prakt.  Chem.  N.  F.  Bd.  vi.  (1872),  S.  1. 

2  Ann.  d.  Ch.  u.  Pharm.  Bd.  xxviii.  (1838),  S.  81. 
'•  Poggendorfs  Annal.  Bd.  lxxxvi.  S.  118. 


20  PEOTEIDS. 

The  product  thus  obtained  is  very  pure,  but  there  is  a  consider- 
able loss  of  material  during  the  washing  of  the  gelatinous  lumps, 
owing  to  the  solubility  of  the  substance  in  the  alkali  which  is 
being  removed.  The  pure  substance  itself  is  also  slightly  solu- 
ble in  water. 

4.     Casein.^ 

This  is  the  well-known  proteid  existing  characteristically  in 
milk  and  in  no  other  fluid  or  secretion  of  the  body.^ 

It  has  recently  been  proposed  to  call  this  proteid  '  caseinogen  '  and 
to  use  the  name  casein  for  the  product  of  its  decomposition,  the  clot 
or  curd,  which  is  formed  by  the  action  of  rennin  upon  it.  This 
nomenclature  would  have  the  advantage  of  indicating  a  relationship 
between  the  two  proteids  similar  to  that  between  fibrin  and  fibri- 
nogen,  myosin  and  myosinogen  (Halliburton). 

Preparation.^  Fresh  milk  is  diluted  with  4  volumes  of  dis- 
tilled water  and  acidulated  with  acetic  acid  until  the  diluted 
milk  contains  from  -075  to  O'l  p.c.  of  the  acid.  If  the  milk  has 
been  diluted  with  ordinary  tap-water  rather  more  acid  must  be 
added.  The  precipitated  casein  is  now  washed  two  or  three  times 
by  decantation  with  water,  as  ra'pidly  as  possible,  dissolved  in  the 
least  quantity  of  dilute  caustic  soda  which  suffices  for  its  solution, 
and  filtered  through  a  series  of  filters  until  the  filtrate  is  quite 
clear  and  only  faintly  opalescent.  This  filtrate  is  then  somewhat 
diluted,  the  casein  again  precipitated  by  the  careful  addition  of 
acetic  acid,  and  the  whole  process  of  washing,  solution,  and  repre- 
cipitation  carried  out  a  second  time.  The  final  product  is  now 
freed  as  far  as  possible  from  water,  worked  up  into  an  emulsion 
with  97  p.c.  alcohol,  collected  on  a  filter,  washed  with  alcohol, 
finally  with  ether,  dried  by  exposure  to  the  air,  and  finally  in  vacuo 
over  sulphuric  acid. 

Casein  may  also  be  separated  from  milk  by  precipitation  with  an 
excess  of  sodium  chloride^  or  magnesium  sulphate.^  The  latter  pro- 
cedure is  chiefly  of  use  for  the  preparation  of  casein  from  human  milk, 
from  which  it  can  scarcely  be  precipitated  by  means  of  acids. 

Pure  casein  as  obtained  by  the  above  method  is  a  fine,  snow- 
white  powder,  which  on  ignition  of  even  large  quantities  of  the 

1  Our  knowledge  of  the  chemistry  and  properties  of  casein  are  hased  chiefly  upon 
the  researches  of  Hammarsten.  His  papers  were  mostly  published  originally  in 
Swedish  or  Latin,  but  are  fully  abstracted  by  himself  in  Medy's  Jahresbencht  d.  Thier- 
cheni.,  to  which  reference  will  in  each  case  be  made. 

'^  For  methods  of  conducting  a  complete  analysis  of  milk  see  Pfeiffer,  Die  Anab/se 
der  Milch,  Wiesbaden,  \S87.  '  ---        ' 

3  Hammarsten,  Maly's  Bericht.  Bd.  tii.  (1877),  S.  1.59. 

*  Hammarsten,  Maly's  Ber.  Bd.  iv.  (1874),  S.  135. 

5  Hoppe-Seyler,  Hdlch.  d.  phys.-path.  chem.  Anal.  Aufl.  iv.  (1875),  S.  241. 


CHEMICAL   BASIS   OF  THE   ANIMAL   BODY.  21 

substance  (4 — 6  grrn.)  leaves  scarcely  a  trace  of  ash.  It  is  prac- 
tically insoluble  in  water,  but  is  soluble  in  alkalis,  carbonates  and 
phosphates  of  the  alkalis,  lime-  and  baryta-water.  From  these 
solutions  it  may  be  precipitated  by  excess  of  neutral  salts  such  as 
sodium  chloride,  and  by  dilute  acids,  in  which  it  is  again  soluble 
if  any  excess  of  acid  is  present.  Its  reactions  thus  correspond 
closely  to  those  of  acid-  and  alkali-albumin,  but  as  will  be  pres- 
ently shewn  it  is  in  many  ways  perfectly  distinct  from  these 
substances.  Solutions  of  pure  casein  are  not  coagulated  by  boil- 
ing, but  if  heated  to  130 — 150°  in  sealed  tubes  a  coagulation  is 
obtained. 

When  acids  are  added  to  diluted  milk  to  effect  the  precipitation  of 
casein  no  precipitate  is  obtained  until  the  solution  has  a  distinctly 
acid  reaction;  this  has  usually  been  attributed  to  the  presence  in  milk 
of  potassium  phosphate.  *  Hammarsten  has  however  shewn  ^  that  the 
same  holds  good  for  solutions  of  casein  free  from  this  salt. 

When  prepared  from  milk  by  magnesium  sulphate  (see  below), 
freed  by  ether  from  fats,  and  dissolved  in  water,  casein  possesses 
a  specific  rotatory  power  (a)D  =  —  80°  ;  in  dilute  alkaline  solu- 
tions, of  -  76°  ;  in  strong  alkaline  solutions,  of  -  91°  ;  in  very 
dilute  solutions,  of  -  87°.^ 

Although  purified  casein  leaves  no  ash-residue  on  ignition,  Ham- 
marsten found  that  it  contained  a  constant  and  fairly  large  amount 
of  phosphorus,  as  a  mean  -847  p.c.  From  this  fact  and  its  be- 
haviour towards  sodium  chloride  in  dilute  solutions,  he  regards 
casein  as  being  a  nucleo-albumin  ^  (see  below).  This  view  cor- 
responds with  the  results  previously  obtained  by  Lubavin,^  who 
found  that  a  phosphorised  (nuclein)  constituent  of  casein  is  sep- 
arated out  as  an  insoluble  residue  during  the  digestion  of  casein 
with  gastric  juice. 

According  to  the  views  of  many  authors  ®  milk  contains  not  one 
casein  only,  but  at  least  two  forms  of  proteid  which  pass  under  the  one 
name.  Hammarsten'^  has  criticised  these  views  and  concludes  that 
casein,  is  a  unitary  substance,  and  not  a  mixture  or  compound. 

Action  of  rennin  on  casein.  This  has  been  fully  studied  by 
Hammarsten,  whose  results  may  be  summarised  as  follows :    Con- 

1  Kiihne,  Lehrb.  d.  phi/siol.  Chem.  1868,  S.  565. 

2  Maly's  Ber.  vii.  S.  162. 

3  Hoppe-Seyler,  Hdbch.  (Ed.  v.)  p,  286. 
*  Maly's  Ber.  iv.  (1874),  S.  1.5.3. 

s  Hoppe-Seyler's  Med.-c/tem.  Untersnch.  Hf.  iv.  (1871),  S,  463. 

s  Millon  u.  Coramaille,  Zt.  f.  Chem.  1865,  S.  641.  Compt.  Rend,  T  i.  n865), 
pp.  118,  859,  T.  II.  p.  221.  Selmi,  Ber.  d.  d.  chem.  Gesell.  Bd.  vii.  (1874),  S  1463. 
Danilewsky  u.  Eadenhausen.  See  Maly's  Ber.  Bd.  x.  (1880),  S.  186.  Zt.  f.  physiol. 
Chem.  Bd.  vii.  (1883),  S.  427.     Struve,  .Tn.  f.  prakt.  Chem.  (2)  Bd.  .^cxix.  S.  71. 

7  Maly's  Ber.  Bd.  v.  (187.5),  S.  119,  Bd.  vi.  (1876),  S.  13,  Zt.f.  physiol.  Chem. 
Bd.  VII.  (1883),  S.  227. 


22  PROTEIDS. 

trary  to  the  older  views  that  the  formation  of  the  clot  is  rather 
of  the  nature  of  a  precipitation  than  a  true  ferment  action,  we 
now  know  that  by  the  action  of  rennin  the  clotting  of  casein  is 
cine  to  a  specific  action  of  the  enzyme  which  results  in  the  form- 
ation of  a  substance  (tyrein)  differing  essentially  from  casein.  It 
had  been  considered  that  the  separation  of  the  clot  was  due  to 
the  formation  of  lactic  acid  from  milk-sugar,^  but  this  is  not  so  ;  ^ 
pure  casein  free  from  every  trace  of  lactic  acid  clots  with  rennin. 
The  specific  action  of  the  enzyme  is  further  shewn  by  the  fact 
that  simultaneously  with  the  formation  of  the  clot,  a  by-product 
is  formed  having  the  properties  of  a  soluble  albumin.^  Further, 
the  clot  is  entirely  different  from  casein :  it  is  much  less  soluble 
in  acids  and  alkalis  than  the  latter,^  always  leaves  as  ordinarily 
prepared  a  large  and  constant  residue  of  ash  (calcium  phosphate) 
on  ignition,  and  even  if  it  be  freed  from  the  calcium  salt  by 
special  methods  ^  and  dissolved  in  dilute  alkalis,  is  not  capable  of 
being  made  to  yield  a  clot  by  the  renewed  action  of  rennin. 

It  may  be  remarked  here  that  no  efforts  to  obtain  a  '  curd '  from 
milk  by  purely  chemical  means,  such  as  the  addition  of  acids  or  neu- 
tral salts,  have  resulted  in  the  jn'oduction  of  a  substance  which  by 
further  treatment  can  be  made  to  yield  a  tyj)ical  ripening  'cheese.' 
The  latter  can  only  be  made  by  the  use  of  rennin. 

The  calcium  salt  plays  an  all-important  part  in  the  clotting  of 
casein.  Casein  freed  from  this  salt  and  dissolved  in  dilute  alkali 
will  not  yield  a  clot ;  dialysed  milk  similarly  yields  no  clot,  but 
if  the  dialysate  be  concentrated  and  added  to  the  milk  it  now 
clots  on  the  addition  of  rennin.  When  pure  casein  is  dissolved  in 
lime-water  and  neutralised  with  phosphoric  acid  it  now  clots  with 
rennin.  The  action  of  the  salt  in  the  whole  process  appears  to  be 
that  it  determines  not  so  much  the  action  of  the  ferment  on  the 
casein,  but  rather  the  subsequent  separation  from  solution  of  the 
altered  product.^  Neither  is  the  calcium  salt  alone  essential,  for 
it  may  be  replaced,  but  with  less  efficient  results,  by  the  similar 
salts  of  magnesium,  barium,  and  strontium.'^ 

The  question  as  to  the  identity  or  the  reverse  of  casein  and 
alkali-albumin  as  obtained  by  the  action  of  alkalis  on  other  pro- 
teids  has  given  rise  to  much  controversy.      Some  authors  have 

1  Soxhlet,  Jn.f.  pr.  Chem.  Bd.  vi.  (1872),  S.  1. 

2  Hanimarsten,  Maly's  Ber.  ii.  (1872),  S.  118,  ia".  (1874),  S.  135.  Heiutz,  Jn.  f 
prakt.  Chem.  N.  F.  Bd.  vi.  (1872),  S.  374. 

3  Hammarsteu.     See  also  Koster  (Swedish)  in  Maly's  Ber.  Bd.  xi.  (1881),  S.  14. 

4  Al.  Schmidt,  BeUr.  z.  Kennt.  d.  Milch,  Dorpat,  1874. 
^  Koster,  loc.  cit.  S.  14. 

*•  For  further  observations  on  the  influence  of  salts  on  the  clotting  of  milk  and 
casein  see  Ringer,  Jl.  of  Physiol.  Vol.  xi.  (1891),  p.  464,  xii.  (1891),  p.  164. 
■  Lundberg  (Swedish).  '  See  Malv's  Ber.  Bd.  vi.  (1876),  S.  11 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.         23 

considered  them  to  be  identical,^  but  that  they  are  not  so  is  suffi- 
ciently shewn  by  the  following  facts.  Solutions  of  alkali-albuuiin 
cannot  be  made  to  clot  by  the  action  of  pure  rennin.  If  milk 
sugar  be  added  to  the  solution  and  im^oure  rennet,  i  e.  extract  of 
the  mucous  membrane  containing  rennin,  be  allowed  to  act  upon 
it,  in  some  cases  a  separation  of  the  alkali -albumin  may  take 
place,  owing  to  the  formation  of  lactic  acid  which  then  precipi- 
tates the  albumin.  In  the  absence  of  the  milk  sugar  no  change 
is  produced  which  can  in  any  way  be  regarded  as  analogous  to 
the  clotting  of  casein.  When  milk  is  clotted  the  separation  of  the 
casein  is  so  complete  that  none  is  found  in  the  '  whey,'  and  Ham- 
marsten  has  shewn  that  if  alkali-albumin  be  added  to  milk  and 
the  mixture  be  then  clotted,  alkali-albumin  may  be  obtained  from 
the  whey  on  breaking  up  the  curd.  It  has  further  been  shewn  ^ 
that  although  casein  is  very  resistant  to  the  action  of  acids,  it 
may  by  treatment  with  them  be  converted  into  acid-albumin  with 
complete  loss  of  all  clotting  powers,  and  still  more  readily  into 
alkali-albumin  by  the  action  of  alkalis. 

A  further  difference  of  the  two  substances  was  urged  by  Zahn  on 
the  basis  of  his  experiments  on  the  filtration  of  milk  through  porous 
earthenware  (battery-cells).^  He  found  that  solutions  of  alkali-albu- 
min pass  through  the  walls  of  the  cells  as  rapidly  as  do  solutions  of 
serum-albumin ;  when  milk  however  is  filtered  by  this  method,  casein 
does  not  pass,  and  the  filtrate  consists  of  water,  salts,  and  the  coagula- 
ble  proteid  of  the  milk.  Whether  this  indicates  any  difference  be- 
tween the  two  substances  is  however  doubtful,  for  it  is  still  an  open 
question  whether  casein  is  truly  in  solution  in  milk.  Further  it  is 
stated  that  the  casein  also  passes  into  the  filtrate  if  the  filtration  is 
prolonged,*  and  Soxhlet  states  that  if  finely  divided  (emulsified)  fat 
be  suspended  in  a  solution  of  alkali-albumin  the  filtration  of  this  sub- 
stance is  rendered  as  impossible  as  that  of  casein  in  milk. 

The  crucial  distinction  between  the  two  substances  is  the  fact 
that  casein  can  be  clotted  by  rennin  with  simultaneous  formation 
of  a  soluble  proteid  by-product,  whereas  no  true  clot  can  ever  be 
obtained  from  ordinary  alkali-alburnin. 

After  the  removal  of  casein  from  milk  by  precipitation,  the 
filtrate  contains  a  small  amount  of  coagulable  proteid,  sometimes 
spoken  of  as  '  lactalbumin,'  closely  resembling  serum-albumin  in 
its  general  properties,  but  differing  slightly  as  to  its  specific 
rotatory  power  and  the  temperature  at  which  it  coagulates  when 
heated.^ 

1  Soxhlet,  loc.  cit. 

-  Lundberg,  loc.  cit. 

3  Zahn,  Pfluger's  Arch.  Bd.  ii.  (1869),  S.  598. 

*  Schwalbe,  Centralh.  f.  d.  med.  Wiss.  1872,  S.  66. 

5  Sebelien,  Zt.  f.  physiol.  Chem.  Bd.  ix.  (1885),  S.  445,  xiii.  (1889),  S.  135.  Eug- 
ling,  see  Maly's  Berickt.  Bd.  xv.  (1885),  S.  183.  Halliburton,  Jl.  of  Physiol.  Vol.  xi. 
(1890),  p.  451. 


24  PEOTEIDS. 

In  addition  to  these,  according  to  the  older  views,  milk,  even 
when  quite  fresh,  frequently  contained  traces  of  a  proteid  which, 
since  it  yielded  the  biuret  reaction,  was  usually  spoken  of  as  a 
peptone,  and  was  by  some  observers  called  '  lactoprotein.^  It 
was  stated  to  increase  in  amount  in  the  milk  on  standing  for  some 
time,  and  more  especially  if  warmed  to  40°,  and  to  be  consider- 
ably increased  during  the  clotting  induced  by  rennin.^  Eecent 
researches  have  however  shewn  that  perfectly  fresh  milk  contains 
no  substance  which  yields  a  biuret  reaction,  its  presence  being  due 
to  its  formation  during  the  processes  employed  in  its  separation.^ 
If  the  milk  undergoes  an  acid  (lactic)  fermentation  a  substance 
may  now  be  obtained  from  it  which  yields  a  biuret  reaction,  but 
is  not  a  true  peptone,  but  a  primary  albumose. 

When  milk  is  kept  for  some  time  at  a  temperature  above  50° 
and  below  its  boiling  point,  a  firm  skin  is  formed  over  its  surface 
composed  largely  of  casein.*  Its  formation  is  n-ot  to  be  regarded 
as  being  specially  characteristic  of  milk,  for  pure  casein  dissolved 
in  dilute  alkalis  exhibits  the  same  phenomenon,  as  also  do  alkali- 
albumin,  chondrin,  gelatin,  and  the  filtrate  from  1  p.c.  starch  when 
it  is  concentrated  on  a  water-bath.  Its  formation  is  probably  due 
to  the  rate  of  evaporation  from  the  surface  of  the  milk  being 
more  rapid  than  the  fluid  diffusion  into  the  upper  layer ;  ^  and  in 
accordance  with  this  it  is  found  that  its  appearance  is  considerably 
facilitated  by  blowing  a  rapid  stream  of  air  or  any  indifferent  gas, 
such  as  carbonic  oxide,  over  the  surface  of  the  warmed  milk. 

Our  knowledge  of  the  chemical  properties  of  casein  as  already 
described  is  based  entirely  upon  researches  carried  out  upon  the 
milk  of  cows.  There  is  no  reason  to  suppose  that  all  that  has 
been  said  does  not  apply  equally  well  to  the  milk  of  other  ani- 
mals. Nevertheless  human  milk  shews,  apart  from  the  difference 
of  composition  (see  §  513),  certain  differences  from  cow's  milk, 
which  are  due  to  a  distinct  but  characteristic  difference  in  the 
reactions  of  the  casein  contained  in  each.^  This  is  shewn  by  the 
following  facts.  (1)  Human  milk  clots  less  firmly  than  cow's 
milk,  and  sometimes  not  at  all  with  rennin.  (2)  The  casein  in 
human  milk,  on  the  addition  of  acetic  acid,  yields  a  very  imper- 
fect precipitate  which  is  finely  flocculent,  almost  granular  as  com- 
pared with  the  compact  and  coarsely  flocculent  precipitate  yielded 


1  Hammarsten,  Maly's  Bericht.  Bd.  vi.  (1876).  S.  13.     Palm  (Russian),  Ibid.  Bd. 
XVI.  (1886),  S.  143.     For  other  references  see  Halliburton,  loc.  cit.  p.  459. 
-  Hoppe-Seyler,  Handbuch  d.  phys.-path.  chem.  Anal.  1883,  S.  480. 
"  Neumister,  Zt.  f.  Biol.  Bd.  xxiv.  (1888),  S.  280. 

4  Sembritzkj^  Pfliiger's  Arch.  Bd.  xxxvii.  (1885),  S.  460.  See  also  Maly's  Ber. 
Bd.  XVII.  (1887),  S.  157. 

5  Hoppe-Seyler,  Virchow's  J rc/i.  Bd.  xvii.  (1859),  S.  420. 

6  Simon,  Animal  Chemistr}!  (Sydenham  Soc),  Vol.  ii.  1846,  p.  53.  Also  in  "Die 
Frauenmilch  u.  s.  w."  Berlin,  1838.  Biedert,  Virchow's  Arch.  Bd.  lx.  (1874),  S. 
352.  Biel,  see  Abst.  in  Maly's  Ber.  Bd.  iv.  (1874),  S.  166.  Langgaard,  Virchow's 
Arch.  Bd.  i.xv.  (1875),  S.  352, 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  25 

by  cow's  milk.  (3)  The  casein  in  human  milk  is,  as  already 
stated,  very  incompletely  precipitated  by  the  addition  of  acids, 
and  can  only  be  completely  precipitated  by  saturation  with  mag- 
nesium sulphate.^  (4)  Casein  from  human  milk  is  less  soluble 
in  water  than  is  that  of  the  cow. 

The  primary  digestive  products  '  caseoses  '  obtained  by  the  action 
of  pepsin  on  casein  have  been  described  and  studied  by  Chittenden 
and  Painter.^ 

Class  III.      Globulins. 

Besides  the  derived  albumins  there  are  a  number  of  native 
proteids  which  differ  from  the  albumins  in  not  being  soluble  in 
distilled  water ;  they  need  for  their  solution  the  presence  of  an 
appreciable,  though  it  may  be  a  small,  quantity  of  a  neutral  saline 
substance  such  as  sodium  chloride.  Thus  they  resemble  the  albu- 
minates in  not  being  soluble  in  distilled  water,  but  differ  from 
them  in  being  soluble  in  dilute  sodium  chloride  or  other  neutral 
saline  solutions.^   Their  general  characters  may  be  stated  as  follows. 

They  are  insoluble  in  water,  soluble  in  dilute  (1  p.c.)  solutions 
of  sodium  chloride;  they  are  also  soluble  in  dilute  acids  and 
alkalis,  being  changed  on  solution  into  acid-  and  alkali-albumin 
respectively  unless  the  acids  and  alkalis  are  exceedingly  dilute 
and  their  action  is  not  prolonged.  The  saturation  with  solid 
sodium  chloride  or  other  neutral  salts  of  their  saline  solutions 
precipitates  most  members  of  this  class. 

1.     Crystallin.     {Glohulin  of  the  crystalline  lens.') 

This  form  of  globulin  is  usually  regarded  as  identical  with 
vitellin.  It  is  however  convenient  to  treat  it  separately,  inasmuch 
as  it  can  be  pre]3ared  in  a  pure  form,  whereas  vitellin  has  not  as 
yet  been  obtained  free  from  lecithin  (see  below). 

Preparation.'^  Crystalline  lenses,  in  which  it  occurs  to  the 
extent  of  24'62  p.c,  are  rubbed  up  in  a  mortar  with  a  little  fine 
sand  and  a  few  crystals  of  rock  salt ;  the  mass  is  then  extracted 
with  water  and  filtered.  The  filtrate  contains  the  crystallin  and 
some  serum-albumin.  The  former  is  separated  from  the  latter  by 
copious  dilution  with  distilled  water  and  passing  a  current  of 
carbonic  anhydride  through  the  diluted  mixture,  whereupon  the 
crystallin  is  precipitated. 

A  dilute  saline   solution  of   this   proteid   coagulates    at   75°. 

1  Makris,  Inaug.-Diss.,  Strassbuig,  1876.    See  Maly's  Ber.  Bd.  vi.  (1876),  S.  113 

2  Stud.  Lab.  Physiol.  Ch.  Yale  Univ.  Vol.  ii.  (1887),  p.  156. 

3  But  see  Nikoljukin  (Russian),  Abst.  in  Maly's  Ber.  Rd.  xviir.  (1888),  S.  5. 

4  Laptschinsky,  Pfliiger's  Arch.  Bd.  xiii.  (187G),  S.  G.31. 


26  PEOTEIDS. 

B^cliamp  has  recorded  ^  some  determinations  of  its  specific  rota- 
tory power  which  must  however  be  accepted  with  caution. 

2.     ViteUin.2 

This  constitutes  the  characteristic  proteid  constituent  of  egg- 
yolk  and  is  also  largely  present  in  caviar.  Some  at  least  of  the 
globulins  present  in  vegetable  protoplasm,  and  more  particularly 
in  the  crystals  of  the  aleurone  grains,  appear  to  be  identical  in 
their  general  properties  and  reactions  with  vitellin.  As  obtained 
in  conjunction  with  some  lecithin  by  exhaustion  of  egg-yolk  with 
ether,  it  consists  of  a  white,  pasty,  granular  mass,  insoluble  in 
water,  readily  soluble  in  solutions  of  sodium  chloride  which  may 
be  easily  filtered.  Unlike  other  true  globulins  it  cannot  be  pre- 
cipitated from  this  solution  by  saturation  with  sodium  chloride. 
Its  saline  solutions  (10  p.c.  NaCl)  are  coagulated  by  heating  to 
75°.  It  is  readily  soluble  in  1  p.c.  sodium  carbonate,  is  incom- 
pletely precipitated  from  this  solution  by  dilution  with  water, 
but  fairly  completely  by  the  additional  passing  of  a  stream  of 
carbonic  acid  gas  through  the  diluted  solution. 

As  has  been  already  stated,  vitellin  is  associated  in  egg-yolk 
with  lecithin  and  (?)  nuclein.  It  has  not  as  yet  been  obtained 
free  from  admixture  with  the  former,  and  a  theory  has  been  ad- 
vanced that  it  is  really  a  complex  substance  resembling  in  this 
respect  haemoglobin,  which  on  treatment  with  alcohol  splits  up 
into  coagulated  proteid  and  lecithin.  It  is  possible  that  pure 
vitellin  free  from  lecithin  might  be  obtained  by  prolonged  ex- 
traction with  ether  in  a  Soxhlet  or  other  form  of  apparatus. 

Fremy  and  Valenciennes  have  described  ^  a  series  of  proteids,  viz. 
ichthin,  ichthidin  &c.  derived  from  the  eggs  of  fishes  and  amphibia. 
They  appear  to  he  closely  related  to  vitellin  but  have  not  been  suffi- 
ciently investigated. 

The  primary  products  obtained  from  vitellin  by  the  digestive 
action  of  pepsin  have  been  examined  and  described  by  Neu- 
meister.* 

Preparation.  Egg-yolk  is  extracted  with  successive  portions 
of  ether  as  long  as  the  residue  yields  any  colour  to  the  solvent. 
The  pasty  residue  thus  obtained  is  dissolved  in  a  minimal  amount 

1  Compt.  Rend.  T.  xc.  (1880),  p.  1255. 

^  Dumas  et  Cahours,  Ann.  Chem.  et  Phys.  (3)  T.  \i.  p.  422.  Hoppe-Seyler,  Med.- 
chem.  Unters.  (Tubingen),  Hft.  2  (1867),  S!  215.  Wevl,  Arch.  f.  Physiol.  Jahrg.  1876, 
S.  546.  Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  635.  Zt.  f.  phi/siol.  Chem.  Bd.  i.  (1877), 
S.  72. 

3   Coinpt.  Rend.  T.  xxxviii.  pp.  469,  525,  570. 

*  Zt.  f.  Biol.  Bd.  XXIII.  (1887),  S.  402.  Cf.  Chittenden  and  Hartwell,  Jl.  of 
Physiol.  Vol.  xi.  (1890),  p.  441. 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.  27 

of  8 — 10  p.c.  sodium  chloride  solution,  precipitated  from  tliis  by 
the  addition  of  an  excess  of  water,  and  purified  by  resolution  in 
the  salt  and  reprecipitation  by  the  addition  of  water.  The  opera- 
tions must  be  conducted  as  rapidly  as  possible  since  the  pro- 
longed action  of  water  renders  the  vitellin  insoluble  in  saline 
solutions.  If  any  attempt  is  made  to  separate  the  vitellin  from 
lecithin  residues  by  means  of  alcohol  it  is  at  once  converted  into 
ordinary  coagulated  proteid. 

3.     Paraglobulin.     (Senwi-globulin.y 

This  proteid  occurs  most  characteristically  in  blood-serum  (also 
in  lymph),  in  amounts  now  known  to  be  much  larger  than  was 
at  one  time  supposed,  and  thus  constituting  about  one-half  of  the 
total  proteids  of  the  serum.^ 

Preparation!^  The  older  methods  consisted  in  (1)  diluting 
serum  ten-fold  with  water  and  passing  a  prolonged  current  of 
carbonic  acid  gas ;  (2)  saturating  serum  with  sodium  chloride. 
The  amount  of  precipitate  thus  obtained  represents  only  a  small 
part  of  the  total  paraglobulin  present  in  the  serum,*  and  the  only 
satisfactory  method  of  preparing  it  pure  and  in  considerable  quan- 
tity is  as  follows :  (3)  serum  is  saturated  at  30°  with  magnesium 
sulphate,  by  means  of  which  paraglobulin  is  quantitatively  pre- 
cipitated. The  precipitate  collected  by  filtration  is  distributed 
through  a  small  volume  of  a  saturated  solution  of  the  magnesium 
salt,  collected  on  a  filter  and  washed  with  saturated  solution  of 
MgS04.  By  this  means  it  is  separated  from  the  larger  part  of 
the  serum-albumin. 

To  effect  its  final  and  complete  separation  from  this  latter  pro- 
teid, two  methods  may  be  adopted,  (a)  The  precipitate  is  dis- 
solved in  water,  then  largely  diluted  and  the  paraglobulin  further 
separated  out  by  passing  a  stream  of  CO2.  (/Q)  The  precipitate 
is  dissolved  as  before  in  water,  the  paraglobulin  again  salted  out 
by  MgS04,  this  process  repeated  several  times,  and  the  final  pro- 
duct separated  from  the  magnesium  salt  by  dialysis.'^ 

1  This  is  the  substance  to  wliich  Al.  Schmidt  gave  the  name  of  fibrino-plastin. 
[Arch.  f.  Anat.  u.  Physiol  Jahrg.  1861,  Sn.  545,  675.  Ibid.  1862,  Sn.  428,  533.  Pflii- 
ger's  Arch.  Bd.  vi.  (1872),  S.  413.  Ibid.  xi.  (1875),  Sn.  291,  526.)  It  had  previously 
been  described  under  the  name  '  serum-casein'  by  Panum.  (Virchow's  Arch.  Bd.  iv. 
(1852),  S.  17.)  The  name  paraglobulin  is  due  to  Kiihne  (Lehrbiich  1868,  Sn.  168, 
175).  It  is  now  generally  and  most  appropriately  known  by  the  latter  name,  or  that 
of  serum-globulin,  as  suggested  by  Hoppe-Seyler. 

-  Hanimarsten,  Pfluger's  Arch.  Bd.  xvii.  (1878),  S.  413,  Salvioli,  Arch.f.  Physiol. 
1881,  S.  269. 

^  Gamgee,  Physiol.  Chem.  Vol.  i.  p.  37. 

*  Hammarsteii,  loc.  cit.     Hevnsius,  Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  549. 

5  Hammersten,  loc.  cit.  Also  Pfluger's  Arch.  Bd.  xviii.  (1878),  S.  38.  Zt.  f. 
physiol.  Chem.  Bd.  viii.  (1883),  S.  467.  Denis  had  previously  used  magnesium  sul- 
phate for  the  quantitative  separation  of  serum-globulins  ("Me'moire  sur  le  Sang, 
1859"),  but  Hammarsten  rediscovered  the  general  method  independently,  and  ap- 


28  PEOTEIDS. 

Pure  paraglobulin  is  insoluble  in  water.  If  dissolved  in  a 
minimal  amount  of  alkali  it  is  precipitated  by  -03  to  -5  p.c.  of 
NaCl.  On  the  addition  of  more  than  -5  p.c.  of  the  salt  it  goes 
again  into  solution  and  does  not  begin  to  be  reprecipitated  on  the 
addition  of  more  salt  until  at  least  20  p.c.  NaCl  has  been  added. 
It  is  not  completely  precipitated  by  saturation  of  its  solutions 
with  NaCl  (Hammarsten).  Its  dilute  saline  solutions  coagulate 
on  heating  to  75°, ^  Dissolved  in  dilute  solutions  of  NaCl  or 
MgS04  its  specific  rotatory  power  is  stated  to  be  (a)D=:  — 47-8°.^ 

Paraglobulin  occurs  in  smaller  amounts  (J — ^)  in  chyle,  lymph, 
and  serous  fluids.  Hammarsten  by  means  of  saturation  with 
MgS04  was  the  first  to  shew  that  hydrocele  fluids  frequently 
contain  paraglobulin,  thus  largely  shaking  the  importance  of  Al. 
Schmidt's  views  as  to  the  part  it  plays  in  the  process  of  blood- 
clotting. 

Globulins  which  are  not  regarded  as  differing  essentially  from 
paraglobulin  are  also  stated  to  occur  in  urine. ^ 

Cell-globulins.  Halliburton  has  described  under  this  name  ^  some 
forms  of  globulin  which  occur  in  lymph-corpuscles  and  may  be  ex- 
tracted from  them  by  solutions  of  sodium-chloride.  Of  these  one,  cell- 
globulin-a,  occurs  in  minute  quantities  only  and  is  characterised  by 
coagulating  at  48-^50°.  The  other,  cell-globulin-/3,  is  more  copiously 
present  in  the  corpuscles  and  coagulates  in  dilute  saline  solutions  at 
75°.  The  latter  resembles  jjaraglobulin  very  closely  in  properties 
other  than  the  identity  of  their  temperatures  of  heat  coagulation  in 
dilute  saline  solution,  e.  g.  precipitability,  &c.  He  considers  that 
cell-globulin-yS  differs  from  true  paraglobulin,  or  plasma-globulin  as 
he  terms  it,  by  possessing  the  power  of  hastening  the  clotting  of  di- 
luted salt-plasma,  and  he  regards  the  so-called  '  fibrin-ferment '  as 
identical  with  cell-globulin-;8  and  arising  from  the  disintegration  of 
leucocytes. 

The  proteid  constituent  of  the  stroma  of  red  blood-corpuscles  con- 
sists chiefly  of  a  globulin  usually  regarded  as  identical  with  paraglo- 
bulin, since  its  saline  solutions  coagulate  at  75°  and  it  is  precipitated 
from  the  same  by  saturation  with  sodium  chloride  and  a  current  of 
carbonic  anhydride.^     Halliburton  considers  it  to  be  identical  with 

plied  it  somewhat  differently  to  Denis.  On  the  use  of  ammonium  sulphate  for 
separating  globulins  and  serum-albumin  see  Michailow  (Russian),  Abst.  in  Maly's 
Bericht.  Bd.  xiv.,  xv.  (1884-5),  Sn.  7,  157.  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd. 
XX.  (1886),  S.  426. 

,1  Halliburton,  Jl.  of  Physiol.  Vol.  v.  (1883),  p.  157. 

2  Fre'dericq,  Arch,  de  Biol.  T.  i.  (1880),  S.  17.  Bull.  Acad.  roy.  de.  Belgique  (2), 
T.  IV.  (1880),  No.  7.     (See  Maly's  Bericht.  1880,  S.  171.) 

3  Lehmann,  Virchow's  Arch.  Bd.  xxxvi.  (1866),  S.  125.  Edlefsen,  Arch.  f.  klin. 
Med.  Bd.  vn.  (1870),  S.  67.  Also  Centralb.  f.  med.  Wiss.  1870,  S.  367.  Senator, 
Virchow's  Arch.  Bd.  lx.  (1874),  S.  476.  Heyusius,  Pfliiger's  Arch.  Bd.  ix.  (1874),  S. 
526  (foot-note).     Fiihry-Snethlage,  Arch.  klin.  Med.  Bd.  xvii.  (1876),  S.  418. 

•»  Proc.  Roy.  Sac.  Vol.  xliv.  (1888),  p.  255.  Ji.  of  Physiol.  Vol.  ix.  (1888). 
p.  235. 

^  Hoppe-Seyler,  Phijsiol.  Chem.  S.  391.  Kuhne,  Lehrhuch,  S.  193.  Wooldridge, 
Arch./.  Physiol.  Jahrg.  1881,  S.  387.  Ho-pjie-SeyleT^  Zt.f.  physiol.  C7ie?n.  Bd.  xm. 
(.1889),  S.  477. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.  29 

cell-globulin-yS,  and  accounts  thus  for  the  earlier  statements  as  to  the 
fibrinoplastic  properties  of  the  stroma-globulins.^ 

4.     Fibrinogen.^ 

This  globulin  occurs  in  blood-plasma  together  with  paraglobu- 
lin  and  serum-albumin.  During  blood-clotting  it  is  converted 
largely,  if  not  entirely,  into  fibrin  (but  see  below).  It  is  also 
found  in  chyle,  serous  fluids  and  transudations,  more  particularly 
in  hydrocele  fluids.^ 

In  its  general  reactions  it  resembles  paraglobulin  but  is 
markedly  distinguished  from  the  latter  by  the  following  charac- 
teristics. (1)  As  it  occurs  in  plasma*  or  in  dilute  solutions  of 
sodium  chloride  (1^ — 5  p.c),  it  coagulates  at  55 — 56°.  (2)  It  is 
very  readily  precipitated  by  the  addition  of  sodium  chloride  to 
its  saline  solutions  until  the  whole  contains  1 6  p.c.  NaCl,  where- 
as paraglobulin  is  not  appreciably  precipitated  until  at  least  20 
p.c.  of  the  sodium  salt  has  been  added. 

Preparation^  Salted  plasma,  obtained  by  centrifugalising  blood 
whose  coagulation  is  prevented  by  the  addition  of  a  certain  pro- 
portion of  magnesium  sulphate,  is  mixed  with  an  equal  volume 
of  a  saturated  (35-87  p.c.  at  14°  C.)^  solution  of  sodium  chloride; 
the  fibrinogen  is  thus  precipitated  while  the  paraglobulin  remains 
in  solution.  The  adhering  plasma  may  be  removed  by  washing 
with  a  solution  of  sodium  chloride,  and  the  fibrinogen  finally 
purified  by  being  several  times  dissolved  in  and  reprecipitated  by 
sodium  chloride. 

Hammarsten's  statements  as  to  the  nature  and  properties  of  fibri- 
nogen have  been  the  subject  of  much  controversy  between  himself,  Al. 
Schmidt,  and  Wooldridge. 

When  a  fluid  containing  purified  fibrinogen  is  made  to  yield 
fibrin  by  the  action  of  fibrin-ferment,  the  amount  of  fibrin  formed 

1  .//.  ofPhi/siol.  Vol.  X.  (1889),  p.  532. 

2  Hammarsten,  Nov.  Act.  Req.  Soc.  Sci.,  TJpsala,  Vol.  x.  1,  1875.  Maly's  Bencht. 
VI.  (1876),  S.  15.  Pfluger's  Arch.  Bd.  xiv.  (1877),  S.  211 ;  xix.  (1879),  S.  563 ;  xxii. 
(1880),  S.  431 ;  xxx.  (1883),  S.  437.  Malv's  Bericht.  xii.  (1882),  S.  11.  Al.  Schmidt, 
Pfluger's  Arch.  Bd.  vi.  (1872),  S.  413;'  xi.  (1875),  S.  291;  xiii.  (1876),  S.  U6. 
"  Lehre  von  den  ferment.  Gerinnunjjserscheinungen  u.  s.  w.,"  Dorpat,  1877.  Wool- 
dridge, Jl.  of  Physiol.  Vol.  IV.  (1883),  pp.  226,  367.  Arch.  f.  Physiol.  1883,  S.  389  ; 
1884,8.313;  1886,  S.  397.  Proc.  Roy.  Sac.  Vol.  lxii.  (1887),  p.  230.  Ludwig  s 
Festschrift,  1887,  S.  221.  Zt.  f.  Biol.  Bd.  xxiv.  1888,  S.  562.  Arch.  f.  Physiol.  1888, 
S.  174.     Jl.  Physiol.  Vol.  x.  (1889),  p.  329. 

3  Hammarsten,  Maly  viii.  (1878),  S.  347. 

*  Pre'de'ricq,  Ann.  Soc.  de  Med.  Gand,  1877.  Arch.  d.  Zool.  Exp.,  1877,  No.  I. 
Bull,  de  VAcad.  roy.  de  Belgique,  T.'LXiv.  (1877),  No.  7.  "Reclierches  sur  la  cod- 
stitution  du  plasma  sanguin."    Paris,  1878. 

s  Hammarsten,  he.  cit.  passim.     Gamgee,  Physiol.  Cfiein.  Vol.  r.  p.  41. 

"  Poggiale,  Ann.  Chim.  Phys.  (3),  Vol.  viii.  p.  469. 


30  PEOTEIODSV 

is  always  less  than  that  of  the  fibrinogen  which  disappears  at  the 
same  time.^  The  deficit  thus  observed  is  at  least  partly  accounted 
for  by  the  simultaneous  appearance  of  a  globulin  which  coagu- 
lates, when  heated  in  saline  solution,  at  64°.  Although  at  first 
sight  it  seems  very  tempting  to  regard  the  process  of  fibrin-for- 
mation from  fibrinogen  as  partaking  of  the  nature  of  a  hydrolytic 
(?)  cleavage  of  which  this  globulin  is  one  product,  this  view  is 
not  as  yet  established.  Hammarsten  considers  it  is  more  prob- 
able that  the  globulin  really  represents  a  portion  of  the  fibrin 
which  has  gone  into  solution  during  its  formation,  basing  his 
views  on  the  earlier  work  of  Denis,^  who  showed  that  under 
special  circumstances  a  form  of  fibrin  may  be  obtained  which  is 
soluble  in  solutions  of  sodium  chloride,  the  solution  coagulating 
at  60 — 65°  (see  below,  p.  33).  Al.  Schmidt  holds  that  Ham- 
marsten's  fibrinogen  as  coagulating  at  55°  is  in  reality  a  sort  of 
modified  or  "  nascent "  fibrin  and  not  truly  a  globulin. 

The  viscid  secretion  of  the  vesicula  seminalis  of  the  guinea-pig  is 
very  rich  in  proteids  and  possesses  the  power  of  clotting.  The  pro- 
teid  which  it  contains  is  not  in  all  respects  a  typical  globulin,  but  in 
many  ways  it  resembles  fibrinogen.  When  dissolved  in  a  little  lime- 
water  it  coagulates  when  heated  to  55°.  The  secretion  itself  clots 
readily  and  firmly  on  the  addition  of  a  small  quantity  of  the  aqueous 
extract  of  a  blood  clot.^ 

The  fibrinogen  of  invertebrate  blood  yields  fibrin  by  the  action  of 
fibrin  ferment,  but  differs  from  vertebrate  fibrinogen  by  coagulating 
at  65°  when  heated.* 

5.     Myosin. 

When  an  irritable  contractile  muscle  passes  into  rigor,  the  sub- 
stance of  which  the  muscle-fibres  are  chiefly  composed  undergoes 
a  change,  analogous  to  the  clotting  of  blood-plasma,  which  results 
in  the  formation  of  a  clot  of  myosin.^  By  appropriate  methods 
(see  §  59)  the  muscle-fibres  may  be  broken  up  and  their  contents 
obtained  as  a  viscid,  slightly  opalescent  fluid  (muscle-plasma), 
which  filters  with  difficulty  and  clots  at  temperatures  above  0°. 
This  muscle-plasma  may  be  diluted  with  solutions  of  varying 
strengths  of  several  neutral  salts,  whereby  its  clotting  may  be 
delayed,  and  the  nature  and  phenomena  of  the  processes  involved 
in  the  clotting  investigated  along  the  lines  previously  employed 
in  the  elucidation  of  the  phenomena  of  the  clotting  of  blood- 

1  Hammarsten,  Pfluger's  Arch.  Bd.  xxx.  (1883),  Sn.  459,  465,  475. 

2  "  Nouvelles  etudes  chimiques,  etc."  Paris,  1856,  p.  106.  "  Memoire  sur  le  sang," 
1859. 

3  Landwehr,  Pfluger's  Arch.  Bd.  xxiii.  (1880),  S.  538. 

4  Halliburton,  Jl.  of  Physiol.  Vol.  vi.  (1884),  p.  321. 

^  Kuhne,  "Das  Protoplasma,"  1864.     Lehrbuch,  S.  272. 


CHEMICAL   BASIS   OF  THE   ANIMAL  BODY.         31 

plasma.i  The  more  important  facts  which  have  thus  been  made 
out  may  be  briefly  summarised  as  follows.  Muscle-plasma  con- 
tains a  globulin-forerunner  of  myosin  ('  myosinogen ')  which 
resembles  fibrinogen  in  coagulating  at  56°.  This  proteid  is  con- 
verted into  myosin  on  the  occurrence  of  clotting  by  the  action  of 
a  specific  ferment,  which  is  regarded  as  being  closely  related  to, 
if  not  identical  with,  an  albumose  (see  below).  The  serum,  which 
is  left  in  small  quantities  only  after  the  formation  of  the  clot, 
contains  proteids  which  coagulate  at  47°^  (paramysinogen) 
63°,  (myoglobulin)  73°,  an  albumin  closely  resembling  serum- 
albumin. 

Preparation.^  (1)  Finely  chopped  muscle- substance  is  washed 
rapidly  with  cold  water,  to  remove  serum-albumin  and  colouring 
matters  (liaemoglobin),  the  residue  is  squeezed  out  in  linen,  and 
extracted  for  at  least  24  hours  with  10  p.c.  solution  of  NH4CI  in 
which  myosin  is  readily  soluble.  The  extract  is  now  filtered  first 
through  muslin  and  then  through  paper;  the  filtrate  is  a  more  or 
less  viscid  and  opalescent  solution  of  myosin.  From  this  the 
myosin  may  be  prepared  in  a  pure  condition  by  allowing  its 
solution  in  the  ammonium  salt  to  drop  into  a  large  excess  of  dis- 
tilled water.  The  myosin  gradually  settles  out  in  a  flocculent 
mass,  which  may  be  further  purified  by  resolution  in  a  minimal 
amount  of  neutral  salt  and  re  precipitation  by  pouring  into  an 
excess  of  distilled  water.  This  purification  must  be  conducted 
rapidly  and  at  low  temperatures,  for  myosin  is  somewhat  readily 
altered  by  the  prolonged  action  of  water  and  becomes  insoluble 
in  saline  solutions.*  (2)  The  finely  chopped  and  washed  muscle 
is  divided  into  two  equal  portions :  to  one  of  these  very  dilute 
(deci-normal)  hydrochloric  acid  is  carefully  added  until  a  distinct 
acid  reaction  is  obtained  as  shewn  by  tropaeolin  00  (see  above, 
p.  18).  The  two  portions  are  then  intimately  mixed  together, 
allowed  to  stand  some  time,  strained  through  muslin,  filtered  and 
the  myosin  precipitated  from  the  filtrate  by  careful  neutralisation 
with  very  dilute  alkali  or  lime-water. 

Apart  from  the  general  reactions  which  characterise  myosin  as 
a  globulin,  it  is  distinguished  by  the  low  temperature  (55 — 56°) 
at  which  its  saline  solutions  constantly  coagulate.  It  leaves  a 
large  ash  residue  on  incineration,  consisting  chieflj!^  of  salts  of 
lime.  As  already  stated,  it  is  converted  into  an  insoluble  proteid 
by  the  prolonged  action  of  water,  and  into  syntonin  by  the  action 
of  acids.  These  substances  are  stated  to  be  capable  of  reconver- 
sion into  myosin  (see  above,  p.  17).     It  is  also  stated^  that  if 

1  Halliburton, .//.  of  Physiol.  Vol.  viii.  (1887),  p.  133. 

2  Cf.  Demant,  Zt.f.  physiol.  Chem.  Bd.  in.  (1879),  S.  241  ;  iv.  (1880),  S.  384. 

3  Danilewsky,  Zt.  f.  pKysiol.  Chem.  Bd.  v.  (1881),  158. 

*  Weyl,  Zt.f.  physiol.  Chem.  Bd.  i.  (1877),  S.  77. 

*  Halliburton,  he.  cit.  p.  148. 


32  PEOTEIDS. 

myosin  is  dissolved  in  NaCl  or  MgS04  (10  and  5  p.c.  respectively) 
it  yields  a  renewed  clot  on  mere  dilution  with  water. 

According  to  Nasse  ^  myosin  constitutes  the  anisotropous  substance 
(see  above  §  56)  of  the  unaltered  inuscle-iibre,  and  the  activity  of  con- 
traction is  inversely  proportional  to  the  amount  of  this  substance 
Avhich  is  present  in  the  fibres  of   different  animals. 

Globulins  to  which  the  name  of  myosin  is  applied  are  described 
as  occurring  in  vegetable  protoplasm  ^  and  in  the  cells  of  the 
liver.^ 

Myosin  is  readily  digested  by  pepsin,  more  slowly  by  trypsin. 
The  primary  products  arising  from  the  digestive  action  of  the 
former  enzyme  have  been  studied  by  Kiihne  and  Chittenden.* 

6.     Globin. 

When  haemoglobin  is  allowed  to  undergo  decomposition  spon- 
taneously by  exposure  to  the  air  an  insoluble  proteid  is  obtained 
of  which  very  little  is  known,  but  to  which  the  name  of  globin 
was  given  by  Preyer.^  It  appears  to  be  perhaps  an  outlying 
member  of  the  globulin  class  of  proteids,  but  unlike  a  true  glob- 
ulin is  scarcely  soluble  in  dilute  acids  and  imperfectly  soluble 
in  alkalis  and  solutions  of  sodium  chloride.  It  is  converted  into 
acid  and  alkali-albumin  by  the  action  of  strong  acids  and  alkalis 
respectively,  and  is  stated  to  yield  no  trace  of  ash  on  incineration 


Class  IV.     Fibrin. 

This  proteid  is  ordinarily  obtained  by  '  whipping '  blood  with 
a  bundle  of  twigs  until  clotting  is  complete ;  the  fibrin  which 
adheres  to  the  twigs  is  then  washed  in  a  current  of  water  until 
all  the  haemoglobin  of  the  entangled  corpuscles  is  removed  and  it 
is  now  quite  white.  The  washing  is  greatly  facilitated  if  the 
fibrin  is  very  finely  chopped  before  it  is  washed,  and  if  it  is  fre- 
quently kneaded  and  squeezed  with  the  hand  during  the  washing. 
In  this  way  it  may  be  obtained  quite  white  in  a  few  hours.  The 
washing  is  also  much  facilitated  if  the  blood  is  mixed  with  an 
equal  bulk  of  water  before  it  is  whipped.  It  is  obvious  that  fibrin 
prepared  by  the  above  method  must  be  in  an  extremely  impure 
condition,  for  it  contains  a  not  inconsiderable  admixture  of  the 

1  "Anat.  u.  Phvsiol.  d.  Muskelsubst."  Leipzig,  1882.  Biol.  Centralh.  Bd,  ii. 
(1882-3),  S.  313.     Zt.  f.  phi/siol.  Chem.  Bd.  vii.  (1882),  S.  124. 

2  Weyl,  Zt.  physioK  Chem.  Bd.  i.  (1877),  S.  96. 

3  Pldsz,  Pfluger's  Arch.  Bd.  vii.  (1873),  S.  377. 

*  Zt.  f.  Biol.  Bd.  XXV.  (1889),  S.  358.  See  also  Cliittendea  and  Goodwin,  Jl.  of 
Physiol.'Yol.  xii.  (1891),  p.  34. 

5  "Die  Blutkrvstalle,"  Jena,  1871,  S.  16G. 


CHEMICAL  BASIS   OF  THE   ANIMAL   BODY.  33 

remains  of  the  white  corpuscles  and  the  stromata  of  the  red.^  It 
can  only  be  prepared  pure  during  the  clotting  of  either  filtered  or 
centrifugalised  iced-plasma  or  salt-plasma,  or  by  the  action  of 
purified  fibrin-ferment  on  pure  fibrinogen.  In  accordance  with 
this,  fibrin  as  ordinarily  obtained  leaves  a  variable  amount  of 
granular  residue  which  contains  phosphorus  during  its  digestion 
by  pepsin.  No  such  residue  is  observed  when  fibrin  from  filtered 
plasma  is  digested  with  pepsin  (see  below,  p.  42),  but  in  no  other 
essential  respect  does  the  one  fibrin  differ  from  the  other. 

Fibrin,  as  ordinarily  obtained,  exhibits  a  filamentous  structure, 
the  component  threads  possessing  an  elasticity  much  greater  than 
that  of  any  other  known  solid  proteid. 

If  allowed  to  form  gradually  in  large  masses,  the  filamentous 
structure  is  not  so  noticeable,  and  it  resembles  in  this  form  pure 
india-rubber.  Such  lumps  of  fibrin  are  capable  of  being  split  in 
any  direction,  and  no  definite  arrangement  of  parallel  bundles  of 
fibres  can  be  made  out. 

Fibrin  is  insoluble  in  water  and  dilute  saline  solutions.  It  is 
also  ordinarily  insoluble  in  dilute  acids  (HCl)  if  their  action 
takes  place  at  ordinary  temperatures  and  is  not  prolonged,  merely 
becoming  swollen  and  transparent  in  the  acid  and  returning  to 
its  original  state  if  the  acid  is  removed  by  an  excess  of  water  or 
careful  addition  of  an  alkali.  By  prolonged  action  at  ordinary 
temperatures,  or  a  shorter  action  at  40°,  the  fibrin  is  profoundly 
changed  and  certain  forerunners  of  the  peptones  which  may  be 
finally  formed  (at  40°)  are  produced  It  is  similarly  insoluble  in 
dilute  alkalis  and  ammonia,  but  passes  more  readily  into  solution 
in  these  reagents,  if  their  action  is  prolonged  or  the  temperature 
is  raised,  than  is  the  case  with  dilute  acids.  The  behaviour  .of 
fibrin  towards  solutions  of  neutral  salts  is  peculiar  and  important. 
As  already  stated,  fibrin  prepared  by  simply  whipping  blood  is 
insoluble  in  dilute  saline  solutions.  But  its  solubility  is  depend- 
ent upon  the  conditions  under  which  it  is  separated  out  from  the 
blood.  In  accordance  with  this,  Denis  ^  described  three  forms  of 
fibrin  to  which  he  gave  the  names  of  1.  Fibrine  concrete  modi- 
fi^e.  2.  Fibrine  globuline.  3.  Fibrine  concrete  pure.  The  first 
is  what  we  now  know  as  ordinary  fibrin  obtained  by  whipping  ar- 
terial blood  (human  in  Denis'  work).  The  second  he  obtained  by 
the  spontaneous  clotting  of  human  venous  blood,  and  this  readily 
swells  up  to  a  slimy  mass  in  10  p.c.  NaCl.  The  third  he  pre- 
pared by  '  whipping '  human  venous  blood  under  certain  precau- 
tions, and  found  it  to  be  soluble  in  dilute  salt  solution  by  one  or 
two  hours'  treatment  with  the  same  at  40°.  Quite  apart  from 
Hammarsten's  partial  confirmation  of  Denis'  statements  there  is 
but  little  reason  for  doubting  the  accuracy  of  so  careful  a  worker. 

1  Hammarsten,  Pfluger's  Arch.  Bd.  xxii.  (1880),  S.  481 ;  xxx.  (1883),  S.  440. 
^  For  reference  see  p.  30. 


34  PROTEIDS. 

The  possible  solubility  of  fibrin  under  certain  conditions  in  saline 
solutions  of  moderate  strength  obtained  considerable  importance 
in  the  controversy  between  Schmidt  and  Hammarsten  as  to  the 
nature  of  the  processes  involved  in  the  clotting  of  blood.  When 
on  the  other  hand  fibrin  is  subjected  to  the  prolonged  action 
of  more  concentrated  (10  p.c.)  solutions  of  neutral  salts,  and 
the  salt  solution  is  frequently  renewed,  the  fibrin  may  be  finally 
completely  dissolved,  being  converted  into  members  of  the  glob- 
ulin class.^  Most  observers  agree  that  the  globulin  thus  chiefly 
formed  coagulates  at  55 — 56°.  Green  obtained  in  addition  one 
coagulating  at  59 — 60°,  the  two  differing  further  in  their  solubili- 
ties in  1  and  10  p.c.  solutions  of  NaCl.  These  changes  are 
brought  about  by  the  salts  in  the  entire  absence  of  any  putre- 
factive phenomena,  and  the  resulting  globulins  cannot  be  made 
to  yield  fibrin  again  by  any  treatment  with  fibrin-ferment. 

When  fresh  unboiled  fibrin  is  simply  washed  till  it  is  white 
and  digested  with  pure  active  trypsin,  it  is  largely  converted 
into  coagulable  proteids  during  the  initial  stages  of  the  ferment 
action.^  These  proteids  are  characteristically  globulins  and  one 
is  closely  related  to  paraglobulin,  as  judged  of  by  its  coagulating 
in  saline  solutions  at  75°  and  possessing  a  specific  rotatory  power 
(in  10  p.c.  NaCl)  of  (a)D= -48-l°.3  The  second  globulin  pro- 
duct of  the  ferment  action  coagulates  at  55 — 56°,  and  in  this 
respect  more  closely  resembles  fibrinogen.^  Whether  the  whole 
of  the  globulin  thus  obtained  is  a  product  of  the  conversion  of 
the  fibrin,  or  whether  a  portion  of  it  is  due  to  globulin  existing  as 
such  in  the  raw  fibrin,  is  not  yet  stated.  Similar  globulins  are 
produced  by  the  action  of  pepsin  in  its  earlier  stages  on  raw 
fibrin.  If  the  fibrin  is  boiled  or  treated  for  some  time  with  al- 
cohol before  digestion  with  either  of  the  above  enzymes,  mere 
traces,  if  any,  of  these  globulins  are  obtained. 

The  purest  fibrin  always  leaves  a  small  but  fairly  constant  ash- 
residue  on  incineration.  Of  the  inorganic  constituents  of  which 
this  residue  is  composed  it  is  probable  that  sulphur  is  the  only 
element  which  enters  essentially  into  the  composition  of  the 
fibrin. 

When  boiled  in  water  or  treated  for  some  time  with  alcohol  it 
loses  its  elasticity,  becomes  much  more  opaque,  is  much  less 
soluble  in  the  various  reagents  which  dissolve  the  original  fibrin 
with  comparative  ease,  is  attacked  with  much  greater  difficulty 

1  Green,  Jl.  of  Physiol  Vol.  viii.  (1887),  p.  373.  Limbourg,  Zt.  f.  physiol.  Chem 
Bd.  xiii.  (1889),  S.  450.  The  latter  contaius  a  complete  list  of  references  to  the 
literature  of  the  subject  excepting  Pldsz,  Pfliiger's  Arch.  Bd.  vii.  (1873),  S.  382. 

2  Brucke,  Wien.  Sitzher.  Bd.  xxxvii.  (18.59).  S.  131.  Kiihne,  Virchow's  Arch.Bd. 
XXXIX.  (1867),  S.  130.  Lehrbuch,  S.  118.  Kistiakowsky,  Pfliiger's  Arch.  Bd.  ix. 
(1874),  S.  446. 

3  Otto,  Zt.  f.  physiol.  Chem.  Bd.  vin.  (1883),  S.  130. 

*  Hasebroek,  Zt.  f.  physiol.  Chem.  Bd.  xi.  (1887),  S.  348.  Herrmann,  Ibid.  S. 
508.  But  see  Neuraeister,  Zt.  f.  Biol.  Bd,  xxiii.  (1887),  S,  398.  Salkowski,  Ibid. 
Bd-  XXV.  (1889),  S.  97. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.  35 

by  pepsin  and  trypsin,  and  is  in  fact  indistinguishable  from  all 
other  coagulated  proteids. 

A  peculiar  property  of  this  body  remains  yet  to  be  mentioned, 
viz.  its  power  of  decomposing  hydrogen  dioxide.  Pieces  of  fibrin 
placed  in  this  fluid,  though  themselves  undergoing  no  change, 
soon  become  covered  with  bubbles  of  oxygen ;  and  guaiacum  is 
turned  blue  by  fibrin  in  presence  of  hydrogen  dioxide  or  ozonised 
turpentine. 

When  globulin,  myosin,  and  fibrin  are  compared  each  with  the 
other,  it  will  be  seen  that  they  form  a  series  in  which  myosin  is 
intermediate  between  globulin  and  fibrin.  Globulin  is  excessively 
soluble  in  even  the  most  dilute  acids  and  alkalis  ;  fibrin  is  almost 
insoluble  in  these ;  while  myosin,  though  more  soluble  than 
fibrin,  is  less  soluble  than  globulin.  Globulin  again  dissolves 
with  the  greatest  ease  in  a  very  dilute  solution  of  sodium  chlo- 
ride. Myosin,  on  the  other  hand,  dissolves  with  difficulty ;  it  is 
much  more  soluble  in  a  10  per  cent,  than  in  a  one  per  cent,  solu- 
tion of  sodium  chloride ;  and  even  in  a  10  per  cent,  solution  the 
myosin  can  hardly  be  said  to  be  dissolved,  so  viscid  is  the  result- 
ing fluid  and  with  such  difficulty  does  it  filter.  Fibrin  again 
dissolves  with  great  difficulty  and  very  slowly  in  even  a  10  per 
cent,  solution  of  sodium  chloride,  and  in  a  one  per  cent,  solution 
it  is  practically  insoluble.  When  it  is  remembered  that  fibrin 
and  myosin  are,  both  of  them,  the  results  of  clotting,  their  simi- 
larity is  intelligible.  Myosin  is  in  fact  a  somewhat  more  soluble 
form  of  fibrin,  deposited  not  in  threads  or  filaments  but  in  clumps 
and  masses. 

Class  V.      Coagulated  Proteids. 

These  are  insoluble  in  water,  dilute  acids  and  alkalis,  and 
neutral  saline  solutions  of  all  strengths.  In  fact  they  are  really 
soluble  only  in  strong  acids  and  strong  alkalis,  though  prolonged 
action  of  even  dilute  acids  and  alkalis  will  effect  some  solution, 
especially  at  high  temperatures.  During  solution  in  strong  acids 
and  alkalis  a  destructive  decomposition  takes  place,  but  some 
amount  of  acid-  or  alkali-albumin  is  always  produced,  together 
with  some  peptone  and  allied  substances. 

Very  little  is  known  of  the  chemical  characteristics  of  this 
class.  They  are  produced  by  heating  to  100°  C.  solutions  of  egg- 
or  serum-albumin  globulins  suspended  in  water  or  dissolved  in 
saline  solutions ;  by  boiling  for  a  short  time  fibrin  suspended  in 
water,  or  precipitated  acid-  and  alkali-albumin  suspended  in 
water.  They  are  readily  converted  at  the  temperature  of  the 
body  into  peptones,  by  the  action  of  gastric  juice  in  an  acid,  or  of 
pancreatic  juice  in  an  alkaline  medium. 

All  proteids  in  solution  are  precipitated  by  an  excess  of  strong 
alcohol.     If  the  precipitant  be  rapidly  removed  they  are  again 


36  FR0TEID8. 

soluble  in  water,  but  if  the  precipitated  proteids  are  .subjected  for 
some  time  to  the  action  of  the  alcohol  they  are,  with  the  excep- 
tion of  peptones,  coagulated  and  lose  their  solubility.  It  appears, 
however,  that  the  proteids  contained  in  the  aleurone-grains  of 
plants  are  exceedingly  resistant  to  this  coagulating  action  of 
alcohol.^ 

Class  VI.     Albtunoses  and  Peptones. 

When  any  of  the  proteids  already  described  are  submitted  to 
the  digestive  action  of  pepsin  or  trypsin,  certain  subtances  are 
formed,  in  the  earlier  stages  of  the  action,  which  are  intermediate 
between  the  proteid  undergoing  digestion  and  the  proteid  product 
(peptone)  which  finally  results  from  the  action  of  the  enzymes. 
When  the  digestive  fluid  employed  is  pepsin  in  presence  of  dilute 
(•2  p.c.)  hydrochloric  acid,  a  small  portion  of  the  proteid  may  be 
at  first  converted  into  a  form  of  ordinary  acid-albumin.^  It  is 
obtained  by  neutralising  a  peptic  digestive  mixture  at  an  early 
stage  of  the  digestion,  and  has  been  frequently  and  almost  usu- 
ally confounded  with  the  '  parapeptone '  of  Meissner.  As  will  be 
explained  later  on,  the  two  substances  are  quite  distinct  forms  of 
proteid.  At  a  later  stage  of  the  digestion  the  first-formed  acid- 
albumin  disappears,  a  considerable  amount  of  parapeptone  is 
formed,  and  other  products  make  their  appearance,  which  are 
known  collectively  under  the  name  of  albumoses.^  By  a  more 
prolonged  action  of  the  pepsin  a  considerable  portion  of  these 
albumoses  is  further  changed  into  the  final  j)roduct  peptones ;  * 
beyond  this  stage  no  further  change  can  be  brought  about  by  the 
action  of  pepsin.  If  tryjDsin  be  employed  in  an  alkaline  solution 
(•25  p.c.  Na.^COs)  the  decomposition  of  the  proteid  is  much  more 
complicated  and  profound.  Instead  of  acid-albumin  a  small  amount 
of  alkali-albumin  makes  its  appearance,  together  with  more  or  less 
(see  above,  p.  34)  of  the  coagulable  globulins  in  the  earliest  stages 
of  the  digestion.  Albumoses  speedily  make  their  appearance,  to 
be  somewhat  rapidly  and  it  may  be  largely  converted  into  pep- 
tones, of  which  some  are  in  their  turn  partially,  though  never 
completely,  converted  into  leucin,  tyrosin,  and  other  less  well- 
defined  crystalline  products.  Similar  products  of  the  decompo- 
sition of  proteids  may  be  obtained  by  the  action  of  acids  alone,  in 

1  Vines,  Jl  of  Physiol.  Vol.  iii.  (1880),  p.  108. 

-  To  this  substance  the  name  '  syntonin '  was  formerly  applied ;  this  term  is  how- 
ever most  appropriately  used  to  denote  that  form  of  acid-albumin  which  results  from 
the  action  of  acids  on  myosin.     (See  above,  p.  16.) 

3  Kuhne,  Verhand.  d.  naturhist.-med.  Ver.  Heidelb.  N.  Y.  Bd.  i.  (1876),  S.  236. 
Schmidt-Miilheim  (Arch.  f.  Phi/siol.  18S0,  S.  36)  named  these  antecedents  of  the 
true  peptones  '  propeptone.'  See  also  Virchow's  Arch.  Bd.  i.  (1880),  S.  575. 
Jahresber.  d.  TRierarzneischule,  Hannover,  1879-1880.  BioI.  Centralb.  Bd.  i. 
(1881-2),  Sn.  312,  341,  558. 

*  Name  due  to  Lehmann  1850,  Physiol.  C/iem.  (Ed.  Cav.  Soc.)  Vol.  ii.  p.  53. 
Peptones  were  first  definitely  described  bv  Mialhe,  Jn.  de  P/iarm.  et  de  Chim. 
(3   Ser.)   T.  x,   1846,   p,    lei." 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.         37 

the  absence  of  all  enzyme,  the  preponderance  of  any  one  or  more 
of  the  products  being  dependent  upon  the  concentration  of  the 
acids,  the  temperature  at  which  they  are  employed,  and  the  dura- 
tion of  their  action.  Proteids  may  also  be  peptonised  by  means 
of  water  acting  at  high  temperatures  under  considerable  pressure. 
By  employing  the  above  means  for  efi'ecting  the  decomposition  of 
proteids,  the  products  (proteid)  which  may  be  obtained,  and  which 
have  of  late  years  been  very  exhaustively  dealt  with  and  described 
by  Klilme  and  his  pupils,  are  numerous.  It  will  hence  conduce  to 
clearness  in  the  subsequent  description  of  each  separate  product 
if  this  is  preceded  by  a  short  statement  of  the  views  which  have 
from  time  to  time  been  held  as  to  the  general  digestive  changes 
which  proteids  may  undergo. 

The  first  distinct  experimental  demonstration  of  the  solvent  action 
of  gastric  juice  was  due  to  Eeaumnr  (1752),  which  was  followed  at 
intervals  by  those  of  Stevens  (1777),  Spallanzani  (1783),  and  Beau- 
mont (1834) .  The  chemical  nature  of  the  products  arising  from  the 
solution  was  not,  however,  described  until  the  year  1846  by  Mialhe 
under  the  name  of  'albuminose;  '  to  these  the  name  of  peptone  was 
subsequently  given  by  Lehmann  in  1850,  and  their  most  important 
properties  fairly  fully  described  by  Mulder  in  1858.  In  this  same 
year  Corvisart  first  published  his  views  as  to  the  specific  proteolytic 
powers  of  pancreatic  juice,  and  these  were  finally  shewn  to  be  correct 
by  Kuhne  in  1867.  During  this  latter  period  (1859 — 1862)  Meissner 
and  his  pupils  ^  had  published  the  results  of  researches  on  the  products 
which  are  formed  during  gastric  digestion.^ 

Meissner' s  researches.  When  an  alkali  was  added  to  the  filtered 
fluid  resulting  from  the  acid  peptic  digestion  of  any  proteid,  to  an 
amount  just  short  of  that  required  for  exact  neutralisation,  a  pre- 
cipitate was  obtained  which  he  named  ijarapeptone.  In  its  gen- 
eral reactions  it  resembled  acid-albumin  or  syntonin,  but  was 
distinctively  characterised  by  its  incapability  of  undergoing  con- 
version into  a  peptone  by  the  further  action  of  pepsin.  He  pointed 
out  at  the  same  time  that  it  might  be  digested  by  an  infusion  of 
the  pancreas.  After  the  removal  of  the  parapeptone  he  occasion- 
ally obtained  a  further  precipitate  by  the  addition  of  acid,  to  not 
more  than  -05  to  •!  p.  c,  to  the  filtrate  ;  this  substance  he  named 
metajtejptone.  He  further  described  a  residue  insoluble  in  dilute 
acids,  but  soluble  in  dilute  alkalis,  which  made  its  appearance 
during  the  digestion  of  casein,  and  to  which  he  gave  the  name  of 
dyspeptone.  After  the  removal  of  the  above  products  there  still 
remained  in  solution  three  substances  called  respectively  a-,  b-, 
and  c-peptone,  and  characterised  as  follows :  — 

a-peptone ;  precipitated  by  strong  nitric  acid  and  by  potassium 
ferrocyanide  in  presence  of  vjeak  acetic  acid. 

1  Zt.f.  rat.  Med.  Bde.  vii.  S.  1 ;  viii.  S.  280 ;   x.  S.  1  ;  xii.  S.  46 ;   xiv.  S.  303. 
^  See  re'sume  by  Lehmann  in  Biol.  Central/).  Bd.  iv.  (1884),  S.  407. 


38  PROTEIDS. 

5-peptone  ,  not  precipitated  by  strong  nitric  acid  nor  by  potas- 
sium ferrocyanide  unless  in  presence  of  an  excess  of  strong  acetic 
acid, 

c-peptone ;  not  precipitated  by  nitric  acid  nor  by  the  potas- 
sium salt,  whatever  be  the  amount  of  acetic  acid  simultaneously 
added. 

These  statements  of  Meissner  led  to  considerable  subsequent 
controversy,  and  the  occurrence  of  the  several  products  he  de- 
scribed was,  with  the  exception  of  parapeptone  and  c-peptone, 
denied  by  those  who  repeated  his  experiments.  There  is  now 
but  slight  reason  for  doubting  that  the  divergent  views  are  due 
to  the  fact  that  Meissner's  digestive  extracts  frequently  contained 
only  small  amounts  of  pepsin,  while  those  of  subsequent  observers 
were  much  more  actively  peptic,  so  that  in  their  case  several  of 
the  intermediate  products  described  by  Meissner  were  rapidly 
peptonised  and  thus  missed.  Further  it  was  urged  that  Meissner's 
parapeptone  was  not  a  specific  product  of  peptic  action,  for  it  was 
said  to  be  identical  in  all  its  chemical  properties  with  ordinary 
acid-albumin  or  syntonin  Hence  it  was  that  Brucke/  opposing 
Meissner,  put  forward  the  view,  which  has  since  been  most  gen- 
erally accepted,  that  the  sole  products  of  a  peptic  digestion  are 
parapeptone  and  peptone,  —  the  former  being  due  to  the  action  of 
the  acid  necessary  for  the  activity  of  the  pepsin,  the  latter  making 
its  appearance  as  the  sole  final  specific  product  of  the  ferment's 
action  on  the  first  formed  parapeptone,  Schiff  alone  appears  to 
have  supported  Meissner.^ 

The  researches  of  Kuhne.  From  what  has  been  already  said  it 
is  at  once  evident  that  Meissner's  view  implied  a  decomposition 
or  splitting-up  of  the  primary  proteid  molecule,  inasmuch  as  he 
held  that  his  parapeptone  was  incapable  of  conversion  into  pep- 
tone by  the  further  action  of  pepsin  Brucke  on  the  other  hand 
regarded  the  process  of  peptonisation  by  gastric  juice  as  not 
necessarily  involving  any  decomposition  of  the  proteid  molecule. 
Kiihne,  impressed  with  the  profound  and  obvious  decomposition 
which  trypsin  brings  about  when  it  acts  on  proteids,  reverted 
once  more  to  the  possibilities  implied  in  Meissner's  views.  In  so 
doing  he  found  further  confirmation  of  the  idea  that  even  in  gas- 
tric peptonisation  the  proteid  is  not  merely  changed  but  split  up, 
in  the  fact  that  only  a  portion  of  the  gastric  peptones  can  be 
made  to  yield  leucin  and  tyrosin  by  the  action  of  trypsin ;  from 
which  it  follows  that  during  a  complete  gastric  peptonisation  at 
least  two  distinct  peptones  are  formed.  In  accordance  with  this 
he  assumed  that  the  original  proteid  molecule  must  itself  consist 
of  two  parts,  of  which  each  yielded   its  corresponding  peptone 

1  Sitzb  d.  Wien.  Akad,  Bd.  xxxvii.  (18.59),  S.  131  ;  xliii.  (1861),  S.  601. 

2  Lecons  sur  la  digestion,  J867,  T.  i.  p,  407  ,  ii.  p.  12. 


CHEMICAL   BASIS   OF   THE  ANIMAL  BODY.  39 

during  the  hydration  which  leads  to  the  formation  of  peptones.^ 
He  found  also  further  confirmation  of  this  probability  in  the  work 
of  Schiitzenberger."^  This  observer,  decomposing  proteids  with 
acids  at  100°  C,  came  to  the  conclusion  that  half  the  proteid 
molecule  is  readily  decomposable  by  the  acids,  while  the  other 
half  is  peculiarly  resistent  and  is  olDtained  in  the  final  products 
as  an  extraordinarily  indigestible  but  true  proteid,  to  which  he 
gave  tlie  characteristic  name  of  '  hemiprotein.'  Convinced  thus 
of  the  double  nature  of  the  proteid  molecule,  and  seeing  but  little 
hope  of  separating  from  each  other  in  a  mixture  the  two  pep- 
tones which  must  presumably  result  from  the  gastric  peptonisa- 
tion  of  a  proteid,  Klihne  endeavoured  to  establish  their  existence 
by  trying  to  discover  the  primary  products  intermediate  between 
the  proteid  and  the  peptones,  —  antipeptone  on  the  one  hand  and 
hemipeptone  on  the  other.^  In  this  his  endeavours  were  at  once 
assisted  by  his  being  in  possession  of  a  large  amount  of  a  proteid 
identical  with  that  first  described  and  carefully  examined  by 
Bence-Jones,  and  hence  called  by  his  name,*  A  renewed  exami- 
nation of  this  substance  revealed  that  it  was  capable  of  con- 
version by  pepsin  into  a  peptone  which  was  readily  further 
decomposed  by  trypsm.^  It  was  in  fact  the  product  intermediate 
between  the  original  proteid  and  the  hemipeptone,  and  to  it 
Kuhne  gave  the  name  of  hemialbumose.  It  now  was  only  neces- 
sary to  obtain  the  corresponding  albumose  precursor  of  the  anti- 
peptone,  to  peptonise  this,  and  shew  that  the  peptone  thus  obtained 
would  yield  no  leucin  or  tyrosin  by  even  prolonged  treatment  with 
trypsin.  This  Klihne  succeeded  in  doing  by  a  fractionated  peptic 
digestion^  and  thus  established  his  own  views,  and  in  doing  so 
shewed  how  accurate  as  a  whole  Meissner's  statements  were. 
This  will  be  evident  from  the  detailed  description  of  the  several 
products  of  the  decomposition  of  proteids  by  pepsin,  trypsin,  and 
acids,  which  is  given  below.  The  fundamental  notion,  then,  of 
Kiihne's  view  is  that  an  ordinary  native  albumin  or  fibrin  con- 
tains within  itself  two  residues,  which  he  calls  respectively  an 
anti-residue  and  a  hemi-residue  The  result  of  either  peptic  or 
tryptic  digestion  is  to  split  up  the  albumin  or  fibrin,  and  to  pro- 
duce on  the  part  of  the  anti-residue  antipeptone,  and  on  the  part 
of  the  hemi-residue  hemipeptone,  the  latter  being  distinguished 
from  the  former  by  its  being  susceptible  of  further  change  by 

1  Verhandl.  d.  naturhist.-med.  Ver.  Heidelberg,  N.  F.  Bd.  i.  (1876),  S.  236. 

2  Bull,  de  la  Soc  chim.  Paris,  T.  xxiii.  (1875),  pp.  161,  193,  216,  242,  385,  433. 
T.  XXIV.  pp.  2,  145.     See  abst,  Maly's  Jahresh.  Bd=  v.  (1875),  S,  299. 

^  The  name  '  hemipeptone  '  was  given  in  order  to  convey  the  idea  that  it  is  the 
peptone  formed  from  one  half  of  the  original  proteid  molecule  '  antipeptone  '  on  the 
other  hand  that  it  is  that  form  of  peptone  which  withstands  or  is  opposed  to  [avri) 
any  further  decomposing  action  of  the  agents  which  led  to  its  appearance- 

*  Phil.  Trans.  Ron.  Soc.  Ft.  i.  1848.  Ann.  d.  Chem.  u.  Pharm.  Bd.  lxvii.  (1884). 
S.  97. 

5  Kuhne,  Zt.  f.  Biol.  Bd.  xix.  (1883),  S,  209. 

6  Kiihne  u.  Chittenden,  Ibid.  S.  171. 


40  PEOTEIDS. 

tryptic  digestion  into  leucin,  tyrosin,  &c.,  each  peptone  being  pre- 
ceded by  a  corresponding  anti-  or  liemi-albumose.  Antipeptone 
remains  as  antipeptone  even  when  placed  under  the  action  of  the 
most  powerful  trypsin,  provided  putrefactive  changes  do  not 
intervene.  Klihne's  views  may  be  conveniently  exhibited  in  the 
accompanying  tabular  forms. 

Decomposition^  of  Proteids  by  Acids. 

1. 

By  -25  p.  c.  HCl  at  40°  C. 

Albumin. 


I  I 

Antialbumate.  Hemialbumose. 


I  I  ^^      I. 

Antialbumid.  Hemipeptoneo     Hemipeptone. 


By  3—5  p.  c.  H2SO4  at  100°  C. 
Albumin. 


Antialbumid.  Hemialbumose. 


I  ^      .    I 

Hemipeptone.      hemipeptone. 

Leucin,  Tyrosin,  etc.    Leucin,  Tyrosin,  etc. 


Decomposition  of  Proteids  by  Digestive  Ferments  (Enzymes). 

Albumin. 


1  ( 


Antialbumose.  Hemialbumose. 


Antipeptone.     Antipeptone.       Hemipeptone.      Hemipeptone. 


Leucin,  Tyrosin,      Leucin,  Tyrosin, 
etc.  etc. 


The  several  products  (antipeptone,  &c.)  are  given  in  duplicate, 
on  the  hypothesis  (of  which  there  is  now  but  little  doubt)  that 
the  changes  of  digestion  are  essentially  hydrolytic  changes,  accom- 
panied by  a  deduplication ;  that  just  as  a  molecule  of  starch  splits 
up  into  at  least  two  molecules  of  dextrose,  or  as  a  molecule  of 


CHEMICAL  BASIS   OF   THE   AMIMAL   BODY.  41 

cane-sugar  splits  up  into  a  molecule  of  dextrose  and  a  molecule 
of  levulose,  so  a  molecule  of  antialbumose,  for  instance,  splits  up 
into  at  least  two  molecules  of  antipeptone,  and  so  on. 

Having  thus  briefly  stated  the  steps  by  which  our  present 
knowledge  has  been  reached  of  the  possible  products  of  a  diges-, 
tive  conversion  of  proteids,  it  now  remains  to  deal  with  these 
products  seriatim.  In  so  doing  it  will  be  best  to  describe  first 
such  products  as  arise  most  largely  and  characteristically  during 
the  action  of  acids,  and  to  treat  of  the  albumoses  and  peptones 
subsequently. 

Antialhumate  This  substance  is,  according  to  Kiihne,  identical 
with  Meissner's  parapeptone.  It  is  most  readily  formed  by  the 
fairly  prolonged  action  of  dilute  acids  at  40°,  but  it  may  also 
make  its  appearance,  but  to  much  smaller  extent,  during  a 
peptic  digestion  in  which  but  little  pepsin  is  present.  It  is  ob- 
tained, mixed  in  some  cases  with  variable  quantities  of  an  ordi- 
nary acid  albumin,  by  neutralising  the  digesting  mixture,  from 
which  it  is  thus  precipitated.  As  already  stated,  it  is  character- 
ised by  the  property  that  it  cannot  be  converted  into  a  peptone 
by  the  most  prolonged  action  of  even  the  most  active  pepsin, 
while  on  the  other  hand  it  is  readily  peptonised  by  trypsin  and 
yields  then  antipeptone,  but  no  leucin  or  tyrosin.  Apart  from 
its  behaviour  with  pepsin  and  trypsin,  it  resembles  ordinary  acid- 
albumin  and  syntonin  in  its  general  chemical  reactions.  But  the 
latter  are  chemically  quite  distinct  from  antialhumate  or  para- 
peptone, for  either  of  them  may  be  peptonised  by  pepsin,  and  the 
peptones  thus  formed  may  be  partly  made  to  yield  leucin  and 
tyrosin  by  the  subsequent  action  of  trypsin. 

Antialhwiid.  By  the  further  prolonged  or  active  treatment  of 
antialhumate  with  acids  it  is  converted  into  the  substance  to  which 
Kiihne  gave  the  name  of  antialbumid.  It  is  in  all  respects  iden- 
tical with  the  '  hemiprotein  '  of  Schiitzenberger,  and  also  probably 
with  the  dyspeptone  of  Meissner.  so  far  as  the  latter  was  not  per- 
haps largely  composed  of  nucleins.  It  also  makes  its  appearance, 
but  in  very  small  amount,  during  a  peptic  digestion,  and  in  con- 
siderable quantity  during  a  pancreatic.  It  is  characterised  by  its 
relatively  great  insolubility  in  dilute  acids  and  alkalis,  so  that  it 
separates  out  as  a  granular  residue  during  a  pancreatic  digestion. 
This  residue  is  readily  soluble  in  1  p.  c.  caustic  soda ;  if  reprecipi- 
tated  by  neutralisation,  it  is  now  soluble  in  1  p.  c.  sodium  car- 
bonate. From  either  of  these  solutions  it  is  very  completely 
precipitated  by  the  addition  of  a  little  sodium  chloride.  In  dilute 
alkaline  solution  (1  p.  c.  Naa  CO3)  it  may  be  partly  converted 
into  a  peptone  by  the  action  of  trypsin,  during  which  process  the 
larger  part  separates  out  into  a  gelatinous  coagulum  or  clot,  which 
is  cj^uite  unacted  upon  by  pepsin  and  can  only  be  peptonised  by 


42  PEOTEIDS. 

the  prolonged  action  of  very  active  trypsin  in  presence  of  a  con- 
siderable amount  (5  p.  c.)  of  sodium  carbonate.  The  peptone 
thus  produced  is  antipeptone,  for  it  yields  no  leucin  or  tyrosin 
by  the  action  of  trypsin. 

It  has  been  suggested  above  that  Meissner's  dyspeptone  might  have 
consisted  largely  of  nuclein,  and  this  possibility  becomes  very  great 
in  the  light  of  the  statements  previously  made  as  to  the  nature  of 
casein  (see  p.  20)  and  the  fact  that  it  was  during  the  digestion  of  this 
proteid  that  he  obtained  the  so-called  dyspeptone.  Even  as  regards 
the  similar  residue  left  during  a  peptic  digestion  of  fibrin,  it  has  been 
stated  that  here  also  the  dyspeptone  is  merely  a  residue  (nucleins) 
from  the  cellular  elements  which  are  ordinarily  entangled  in  the 
fibrin;  in  support  of  this  it  is  stated  that  no  dyspeptone  is  obtained 
during  the  digestion  of  fibrin  prepared  from  filtered  plasma.^  There 
is  however  now  no  doubt  from  Ktihne's  researches  that  anti-albumid 
is  a  true  proteid,  not  a  mere  undigested  residue  of  nucleins,  and  that 
its  properties  are  generally  such  as  Meissner  described  for  his  dys- 
peptone. 

The  albumoses.  These  are  the  true  primary  products  of  the 
action  of  the  proteolytic  enzymes  on  proteids,  and  give  rise  by  the 
further  action  of  the  ferments  to  the  corresponding  peptones.  In 
accordance  with  Kiihne's  views  already  stated  there  must  of  ne- 
cessity be  at  least  two  albumoses,  antialbumose  the  forerunner  of 
antipeptone,  and  hemialbumose  of  hemipeptone. 

Antialhumose}  This  substance  is  obtained  as  a  neutralisation 
precipitate  at  a  certain  early  stage  of  a  fractionated  peptic  diges- 
tion of  proteids.  In  its  ordinary  chemical  reactions  it  is  indis- 
tinguishable from  acid-albumin  or  syntonin.  It  may  be  converted 
into  a  peptone  by  the  further  action  of  pepsin,  and  still  more 
readily  by  the  action  of  trypsin,  so  that  it  does  not  make  its  ap- 
pearance in  the  final  products  of  either  a  prolonged  peptic  or  a 
short  tryptic  digestion.  The  peptone,  into  which  it  may  be  con- 
verted by  either  pepsin  or  trypsin,  is  antipeptone,  for  it  cannot 
be  made  to  yield  any  trace  of  leucin  or  tyrosin  by  even  the  most 
prolonged  and  energetic  treatment  with  trypsin,  and  in  this  fact 
lies  the  distinction  between  antialbumose  and  either  acid-albumin 
or  syntonin.  During  its  peptonisation  by  trypsin  some  antialbu- 
mid  is  simultaneously  formed.  Antialbumose  differs  from  para- 
peptone  by  the  fact  that  the  latter  can  only  be  peptonised  by 
trypsin,  the  former  by  either  pepsin  or  trypsin. 

Hemialhumose?'     This  is  the  best  known,  most  characteristic 

1  Hammarsten,  Pfliiger's  Arch.  Bd.  xxx.  (1883),  S.  440. 

2  Kiihne  a.  Chittenden,  Zt.f.  Biol.  xix.  (1883),  Sn.  170,  194. 

3  Schmidt-Miilheim,  antea  'loc.  cit.  Salkowski,  Virchow's  Arch.  Bd.  81.  (1880), 
S.  552.  Kiihne  and  Chittenden,  Joe.  cit.  and  Zt.  f.  Biol.  Bd.  xx.  (1884),  S.  11. 
Herth,  Monatshefte  f.   Chem.  Bd.  v.  (1884),  S.  266.     Straub  (Dutch).      See  Maly's 


CHEMICAL  BASIS   OF   THE  ANIMAL   BODY.  43 

and  most  frequently  obtained  by-product  of  proteid  zymolysis.^ 
It  was  first  noticed  and  isolated  by  Meissner  under  the  name  of 
a-peptonCj  is  identical  with  Bence-Jones'  proteid  in  the  urine  of 
osteomalacia,  and  has  also  been  known  under  the  name  of  'pro- 
peptone.'  Of  late  years  it  has  been  recognised  as  occurring  not 
infrequently  in  urine,^  and  it  is  more  than  probable  that  many  of 
the  older  statements  as  to  the  occurrence  of  peptones  in  urine 
and  other  fluids  referred  really  to  the  occurrence  of  hemialbu- 
mose.  It  is  also  stated  to  occur  normally  in  the  marrow  of 
bones,^  and  in  cerebrospinal  fluid.*  Since  it  is  readily  peptonised 
by  trypsin  with  the  simultaneous  formation  from  the  peptone  of 
much  leucin  and  tyrosin,  hemialbumose  scarcely  makes  its  ap- 
pearance in  any  appreciable  quantity  in  the  final  products  of  a 
pancreatic  digestion.  It  is  best  prepared  by  the  action  of  a  small 
amount  of  very  active  pepsin  on  a  considerable  mass  of  fibrin, 
previously  swelled  up  into  a  gelatinous  mass  by  the  action  of 
•2  p.c.  HCl  at  40°  ^.  Under  the  action  of  the  pepsin  the  fibrin 
liquefies ;  as  soon  as  this  is  complete,  dilute  sodium  carbonate  is 
added  until  the  reaction  is  just  faintly  alkaline,  by  which  means 
a  bulky  precipitate  is  obtained.  This  is  removed  by  filtration  and 
the  filtrate  now  contains  a  large  amount  of  hemialbumose  and  but 
little  peptone,  and  may  be  utilised  directly  for  the  tests  charac- 
teristic of  the  albumose. 

Preijaration  of  imre  hemialbumose  (Salkowski).^  Acidulate  the 
filtrate  described  above  strongly  with  acetic  acid,  add  an  excess 
(37-5  grms.  to  each  100  c.c.)  of  sodium  chloride,  and  agitate  the 
mixture  until  it  is  saturated  with  salt.  The  hemialbumose  is 
thus  precipitated;  it  is  now  collected  on  a  filter,  washed  with 
saturated  solution  of  sodium  chloride,  dissolved  again  in  water, 
and  reprecipitated  by  acetic  acid  and  sodium  chloride.  This 
process  is  repeated,  and  the  final  product  is  then  dissolved  in  a 
minimal  amount  of  water  and  freed  from  salt  by  dialysis.''  It 
may  then  be  concentrated,  precipitated  by  alcohol  and  dried,  first 
over  sulphuric  acid  and  then  at  105°. 

Reactions  of  hemialbumose.  The  pure  dry  substance  is  not 
readily  soluble  in  distilled  water,  but  readily  soluble  in  traces  of 

Bericht.  Bd.  xiv.  (1884),  S.  28.  Hamburger  (Dutch).  See  Malv's  Bericht.  Bd.  xvi. 
(1886),  S.  20. 

^  This  expression  may  be  conveniently  used  to  denote  generally  the  change? 
produced  by  the  unorganised  ferments. 

2  Salkowski  u.  Leube,  '  Die  Lehre  von  Harn.'     1882,  Sn.  210,  350. 

3  Fleischer,  Virchow's  Arch.  Bd.  81  (1880),  S.  188. 
*  Hallibiirton,  Jl.  of  P/njsiol.  Vol.  x.  (1889),  p.  232. 

5  For  precise  details  see  Zt.f.  Biol.  Bd.  xix.  (1883),  S.  184.  See  also  Drechsel, 
"  Anleituug  zur  Darstell.  physiol.-chem.  Praparate."     Wiesbaden,  1889,  S.  23. 

6  Virchmv's  Arch.  Bd.  lxxxi.  (1880),  S.  552. 

"^  During  the  dialysis  some  loss  of  albumoses  occurs,  since  they  are  slightly  dif- 
fusible, but  less  so  than  the  peptones.     Zt.f.  Biol.,  Bd.  xx.  (1884)  "Note  on  p.  27. 


44  PROTEIDS. 

acids,  alkalis,  and  ueutral  salts  (sodium  chloride).     These  solu- 
tions give  the  following  characteristic  reactions :  — 

1.  Acidulate  fairly  strongly  with  acetic  acid  and  add  a  few 
drops  of  saturated  solution  of  sodium  chloride  ;  a  precipitate  is 
formed  which  disappears  on  warming  and  comes  down  again  on 
cooling.  If  excess  of  the  salt  is  added  the  precipitate  does  not 
dissolve  on  warming. 

2.  Add  carefully  a  few  drops  of  pure  nitric  acid  ;  a  precipitate 
is  formed  if  the  acid  is  not  in  excess,  which  disappears  on  warm- 
ing and  comes  again  on  cooling. 

3.  Add  acetic  acid,  avoiding  all  excess,  and  then  a  trace  of 
potassium  ferrocyanide  ;  a  precipitate  is  formed  which  disappears 
on  warming  and  reappears  on  cooling. 

4.  On  the  addition  of  caustic  soda  in  excess  and  a  trace  of 
sulphate  of  copper  the  ordinary  biuret  reaction  is  obtained.  This 
reaction  distinguishes  hemialbumose  from  other  soluble  proteids, 
with  the  exception  of  peptones. 

Hemialbumose  has  so  far  been  spoken  of  as  being  one  uniform 
substance  only.  Kiihne  and  Chittenden  in  their  earlier  work  ^  at 
first  distinguished  merely  between  a  soluble  and  insoluble  form  ; 
more  recently  they  have  described  four  closely  allied,  but  distinct 
forms  of  the  albumose.^  (1)  Protalbumose.  Soluble  in  hot  and 
cold  water  and  precipi table  by  NaCl  in  excess.  (2)  Deuteroal- 
humose.  Soluble  in  water,  not  precipitated  by  NaCl  in  excess, 
unless  an  acid  be  added  at  the  same  time.  (3)  Heteroalbitmose, 
Insoluble  in  hot  or  cold  water ;  soluble  in  dilute  or  more  concen- 
trated solutions  of  sodium  chloride,  and  precipitable  from  these 
by  excess  of  the  salt.  (4)  Dysalhumose.  Same  as  heteroalbumose, 
except  that  it  is  insoluble  in  salt  solutions.^  Hemialbumose  as 
ordinarily  prepared  may  hence  be  regarded  as  a  mixture  of  these 
several  albumoses  in  varying  proportions  according  to  the  condi- 
tions of  its  preparation. 

The  preceding  statements  as  to  the  existence  of  four  forms  of 
hemialbumose  are  however  contested  by  Herth,  Straub,  and  Ham- 
burger (loc.  cit.  on  p.  42). 

The  peptones.  Eecent  work  has  shewn  that  in  all  probabil- 
ity the  various  substances  which  have  been  described  as  peptones 
have  consisted  to  some  extent,  if  not  largely,  of  a  mixture  of  true 
peptones  with  variable  quantities  of  albumoses.  Our  knowledge 
of  the  nature  and  properties  of  true  peptones  is  at  present  in  a 

1  Zt.  f.  Biol.  Bd.  XIX,  (1883),  T.  174. 

2  /W.  Bd-.  XX-  (1884),  S.  11, 

3  For  further  details  the  original  papers  of  Kiihne  and  Chittenden  must  he  con- 
sulted,more  especially  Zt.  f.  Biol.  Bd.  xx.  (1884),  S.  11.  See  also  Neuraeister,  Zt  f. 
Biol.  Bde.  xxiii-  (1887),  S."381  ;  xxiv.  (1888),  S.  267 ;  xxvi.  S  324.  The  preparation 
and  separation  of  the  albumoses  is  conveniently  given  in  Rohmann's  "  Anleitung  znm 
chemischen  Arbeiten"     Berlin,  1890,  S.  48. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.  45 

state  of  transition,  so  tliat  it  is  on  the  whole  advisable  to  give 
some  account  of  the  older  work  as  well  as  of  the  more  recent. 

Preparation  of  'peptoms.  For  this  the  works  of  Maly,^  Herth,^ 
Henninger,^  KosseL^  Hofmeister,^  and  Low^  should  be  consulted. 
The  general  properties  and  reactions  of  the  peptones  obtained  by 
the  above  authors  may  be  stated  as  follows.  As  precipitated  by 
alcohol  they  consist  of  a  white  or  yellowish  powder,  which  is 
hygroscopic  and  extraordinarily  soluble  in  water,  and  in  some 
cases  may  even  be  deliquescent.  Unless  thoroughly  dehydrated 
the  powder  may  melt  on  gentle  warming.  From  their  neutral 
aqueous  solutions  they  are  precipitated  with  difficulty  by  a  large 
excess  of  alcohol,  being  unchanged  in  the  process  and  not  becom- 
ing coagulated  or  insoluble  by  prolonged  exposure  to  the  action  of 
the  precipitant.  The  precipitation  occurs  with  difficulty  if  at  all 
in  presence  of  hydrochloric  acid,  Peptones  are  not  precipitated 
by  many  of  the  reagents  which  precipitate  other  proteids,  but  are 
precipitated  by  tannic  acid,  mercuric  chloride,  nitrates  of  mercury, 
and  by  phosphotungstic  and  phosphomolybdic  acids  in  presence  of 
hydrochloric  or  other  mineral  acids ;  also  by  the  double  iodides  of 
potassium  and  mercury  or  potassium  and  bismuth,  in  presence 
of  strong  mineral  acids.  A  very  characteristic  reaction  is  the 
'  biuret '  or  pirik  coloration  which  is  obtained  on  the  addition  of 
an  exce&s  of  caustic  soda  and  a  mere,  trace  of  sxilphate  of  copper. 
The  slightest  excess  of  the  copper  salt  gives  a  violet  colour,  as 
is  the  case  with  all  other  proteids,  which  deepens  in  tint  on  boil= 
ing.  This  biuret  reaction  is  however  now  known  to  be  yielded 
also  by  the  albumoses  (see  above).  Peptones  are  all  laevorotatory 
and  diffusible. 

The  diffusihility  of  peptones  is  relatively  great  iii  comparison  with 
that  of  other  forms  of  proteids  ;  it  is  however  absolutely  small  when 
compared  with  that  of  crystalline  substances  such  as  sodium  chloride, 
and  hence  they  may  be  separated  from  admixed  salts  by  dialysis.  All 
statements  as  to  their  absolute  diffusibility,  as  based  on  earlier  state- 
ments, must  however  be  received  with  caution,  in  view  of  the  transi- 
tional state  of  our  information  as  to  the  properties  of  the  true  peptones. 

Of  late  years  it  has  been  observed  that  the  complete  separation 
of  peptones  from  albumoses  is  possible  by  taking  advantage  of  the 
fact  that  the  latter  are  all  completely  precipitable  by  saturation 
with  neutral  ammonium  sulphate,  whereas  the  former  are  not." 
By  means  of  this  difference  in  the  behaviour  of  the  two  classes  of 

1  Pfliiger's  Arch.  Bd.  ix.  (1874),  S.  585. 

2  Zi.  f.  physiol.  Chem.  Bd.  i,  (1877),  S.  273. 

^  "  De  la  nature  et  du  role  physioloe;ique  des  Peptones."     Paris,  1878. 

4  Zt.  f.  physiol.  Chem.  Bd.  ni.  (1879"),  S.  58. 

5  Ibid.  Bd.  V.  (1881).  S.  129. 

6  Pflu£>-er's  Arch.  Bd.  xxxi.  (1883),  S.  408. 

■^  Wenz,  Zt.  f.  Bwl.  Bd.  xxii.  (1886),  S.  10.  Kuhne,  Verhandl.  d.  naturhist.-med. 
Ver.  Heidelberg,  Bd.  iii-  (1885),  S,  286. 


46  PKOTEIDS. 

substances  to  the  ammonium  salt,  Kiihne  and  Chittenden  have 
prepared  what  they  regard  as  the  true  pure  peptones  as  follows.^ 
The  products  of  a  digestion  are  neutralised,  filtered,  very  faintly 
acidulated  with  acetic  acid  and  saturated  with  the  ammonium 
salt.  The  filtrate  from  the  precipitate  thus  obtained  is  largely 
freed  from  the  excess  of  salt  by  careful  concentration  on  a  water- 
bath.  The  ammonium  salt  is  then  got  rid  of  by  the  addition  of 
baryta  water  and  barium  carbonate  in  slight  excess,  and  after 
filtration  these  reagents  are  finally  removed  by  the  careful  addi- 
tion of  dilute  sulphuric  acid.  The  peptone  thus  obtained  may  be 
still  further  purified  by  precipitation  with  phosphotungstic  acid.^ 
The  pure  peptones  thus  prepared  are  strikingly  non-precipitable 
by  many  of  the  reagents  by  which  other  proteids  may  be  precipi- 
tated, more  especially  by  ferrocyanide  of  potassium  in  presence 
of  acetic  acid,  a  reagent  by  which  practically  all  other  proteids 
in  solution  are  precipitated.  No  quantitative  statements  have  as 
yet  been  made  as  to  their  rotatory  power  or  diffusibility.  They 
are  stated  to  have  such  an  affinity  for  water  that  a  small  portion 
of  the  dry  substance  when  moistened  with  water  exhibits  the 
same  phenomena  as  does  phosphoric  anhydride  under  similar  con- 
ditions. They  also  yield  an  intense  '  biuret '  reaction  with  caustic 
soda  and  sulphate  of  copper. 

Antipeptone  may  be  obtained  by  the  action  of  either  pepsin  or 
trypsin  on  antialbumose,  or  by  the  action  of  trypsin  on  antialbumate 
or  antialbumid.  When  purified  no  leucin  or  tyrosin  can  be  obtained 
by  the  most  prolonged  action  of  trypsin  on  this  peptone. 

Remipieptone  is  best  obtained  by  the  action  of  pepsin  on  hemi- 
albumose.  When  purified  and  digested  with  trypsin  it  yields  much 
leucin  and  tyrosin,  and  in  this  respect  alone  does  it  differ  from 
antipeptone. 

Amphopeptone.  This  is  the  mixture  of  anti-  and  hemi-peptone  re- 
sulting from  the  action  of  pepsin  on  proteids. 

Notwithstanding  the  probable  formation  of  peptones  in  large 
quantities  in  the  stomach  and  intestine,  to  judge  from  the  results 
of  artificial  digestion,  a  very  small  quantity  only  can  be  found  in 
the  contents  of  these  organs.^  They  are  probably  absorbed  as 
soon  as  formed.  Another  point  of  interest  is  their  reconversion  into 
other  forms  of  proteids,  since  this  must  occur  to  a  great  extent  in 
the  body.  We  are  however  as  yet  ignorant  of  the  manner  in  which 
this  reverse  change  is  effected. 

It  is  now  generally  considered  that  the  peptones  are  products 
of  the  hydrolytic  decomposition  of  the  proteids  from  which  they 
are  formed.  This  view  is  based  partly  upon  general  considera- 
tions as  to  the  probable  nature  of  the  change,  from  observations 

1  Zt.  f.  Biol.  Bd.  XXII.  (1886),  S.  423. 

2  Hirschler,  Zt.  f.  physiol.  Chem.  Bd.  xi.  (1887),  S.  28.  Otto,  Ibid.  Bd.  viii. 
(1883),  S.  136. 

3  Schmidt-Miilheim,  Arch.   f.  Physiol.  1879,  S.  39. 


CHEMICAL  BASIS   OF   THE  ANIMAL   BODY.  47 

of  the  conditions  under  which  they  are  formed,  and  which  are 
known  to  be  hydrolytic  in  other  cases,  e.  g.  the  conversion  of 
starch  into  sugar  by  the  action  of  enzymes  and  acids.  There  is 
further  a  certain  amount  of  direct  evidence  that  their  formation 
is  accompanied  by  the  assumption  of  water.^  Finally  there  is  an 
increasing  amount  of  evidence,  based  on  analyses  of  proteids  and 
the  peptones  which  may  be  formed  from  them,  that  the  latter 
contain  less  carbon,  i.  e.  more  hydrogen  (?)  and  oxygen  than  the 
former.^  But  this  latter  evidence  is  as  yet  merely  suggestive. 
It  is  however  borne  out  by  analysis  of  gelatin-peptones.'^  The 
one  important  fact  in  connection  with  the  relationship  of  the 
peptones  to  the  mother  proteids  is  that  they  are,  as  already 
stated,  products  of  the  decomposition  of  the  latter  and  of 
smaller  molecular  weight,  an  assumption  which  is  warranted 
not  only  by  the  whole  tendency  of  recent  investigation,  but 
more  especially  by  the  fact  that  whereas  ordinary  proteids  are . 
non-diffusible,  peptones,  and  to  a  less  degree  the  albumoses, 
are  diffusible. 

According  to  the  views  of  some  observers  it  is  said  to  be  pos- 
sible to  effect  a  partial  reconversion  of  peptones  into  the  more 
primary  proteids  from  which  they  were  obtained,  by  means  of 
prolonged  heating  to  140 — 170°,  and  possibly  by  means'  of  a 
dehydrating  agent  such  as  acetic  anhydride.*  But  little  is  how- 
ever definitely  known  as  to  the  real  nature  of  the  products  ob- 
tained by  these  means. 

It  was  at  one  time  stated  that  when  peptones  are  injected  into 
the  blood-vessels,  the  blood  speedily  loses  its  power  of  clotting 
after  removal  from  the  body.^  This  action  is  now  known  to  be 
due  to  the  albumoses  with  which  the  peptones  were  mixed.^  The 
clotting  may  similarly  be  prevented  by  the  injection  of  a  1  p.c. 
NaOl  extract  of  the  pharynx  and  gullet  of  the  leech :  the  cause 
of  this  has  not  as  yet  been  fully  worked  outJ 

During  the  pancreatic  digestion  of  proteids  some  by-product  makes 
its  appearance  which  gives  a  characteristic  violet  or  pink  coloration  on 
the  addition  of  bromine,  or  of  chlorine  in  the  presence  of  acetic  acid. 

1  Danilewski,  Centralh.  f.  d.  mecl  Wiss.  1880,  Nr.  42  ;  1881,  Nr.  4  u.  5.  Arch.  d. 
Sci.  ph)/s.  et  nat.  T.  vii.  (1883),  p.  150,  425. 

'-  Otto,  he.  cit.     Klihne  u.  Chittenden,  Zt.  f.   Biol.  xix.  203  ;  xxii.  452. 

3  Tatarinoff,  Compt.  Rend.  T.  97.  (1883),  p.  713.  Hofmeister,  Zt.  f.  phijsiol. 
Chem.  Bd.  ii.  (1878),  S.  299.  Klug,  Pfliiger's  Arch.  Bd.  xlviii.  (1890),  S.  100 
But  see  also  Chittenden  and  Solley, .//.  of  Physiol.  Vol.  xii.  (1891),  p.  33,  on  the 
gelatoses. 

*  Henninger,  loc.  cit.  on  p.  45.  Hofmeister,  Zt.  f.  physiol.  Chem.  Bd.  ii.  S.  206. 
Pekelharing,  Pfliiger's  Arch.  Bd.  xxii.  (1880),  S.  196.  Kiihne,  Verhdnd.d.  naturhist.- 
med.  Ver.,  Heidelberg,  Bd.  iii.  (1885),  S.  290.  Neumeister,  Zt.  f.  Biol.  Bd.  xxiii. 
<1887),  S.  .394. 

a  Schmidt-Miilheim.  Arch.  f.  Physiol.  1880,  S.  33.     Fano,  Ibid.  1881,  S.  277. 

6  PoUitzer,  ,//.  of  Physiol.  Vol.  vir,  (1885),  p.  283. 

■^  Haycraft,  A-oc.  Roy.  Sac.  No.  231,  1884.  Arch.  f.  exp.  Path.  u.  Pharm.  Bd. 
xvjii.  (1884),  S.  209.     Dickinson,  .//.  oj  Physiol.  Vol.  xi.  (1890),  p.  566. 


48  PEOTEIDS. 

The  colour  is  not  due  to  the  peptones  or  albumoses  (Klihne).  The 
colouring  matter  obtained  by  the  addition  of  these  reagents  has  been 
examined  b}'  Krukenberg  ^  and  more  recently  by  Stadelmann.'-^ 

Class  VII.     Lardacein,  or  the  so-called  amyloid  substance.^ 

The  substance,  to  which  the  above  name  is  applied,  is  found  as 
a  pathological  deposit  in  the  spleen  and  liver,  also  in  numerous 
other  organs,  such  as  the  blood-vessels,  kidneys,  lungs,  &c. 

It  is  insoluble  in  water,  dilute  acids  and  alkalis,  and  neutral 
saline  solutions. 

In  percentage  composition  it  is  almost  identical  with  other 
proteids,*  viz. :  — 

0.  and  S.  H.  K  C. 

24-4  7-0  15-0  53-6 

The  sulphur  in  this  body  exists  in  the  oxidised  state,  for  boil- 
ing with  caustic  potash  gives  no  sulphide  of  the  alkali.  The 
above  results  of  analysis  would  lead  at  once  to  the  ranking  of 
lardacein  as  a  proteid,  and  this  is  strongly  supported  by  other 
facts.  Strong  hydrochloric  acid  converts  it  into  acid-albumin, 
and  caustic  alkalis  into  alkali-albumin.  When  boiled  with  dilute 
sulphuric  acid  it  yields  leucin  and  tyrosin  ;  ^  by  prolonged  putre- 
faction indol,  phenol,  &c.  ^  On  the  other  hand,  it  exhibits  the 
following  marked  differences  from  other  proteids : — It  wholly 
resists  the  action  of  ordinary  digestive  fluids ;  it  is  coloured  red, 
not  yellow,  by  iodine,  and  violet  or  pure  blue  by  the  joint  action 
of  iodine  and  sulphuric  acid.  From  these  last  reactions  it  has 
derived  one  of  its  names,  '  amyloid,'  though  this  is  evidently  badly 
chosen ;  for  not  only  does  it  differ  from  the  starch  group  in  com- 
position, but  by  no  means  can  it  be  made  to  yield  sugar : "'  this 
latter  is  one  of  the  crucial  tests  for  a  true  member  of  the  carbo- 
hydrate group.  According  to  Heschl  ^  and  Cornil  ^  anilin-violet 
(methyl-anilin)  colours  lardaceous  tissue  rosy  red,  but  sound 
tissue  blue. 

The  colours  mentioned  above,  as  being  produced  by  iodine  and 
sulphuric  acid,  are  much  clearer  and  brighter  Avhen  the  reagents  are 
applied  to  the  purified  lardacein.  When  the  reagents  are  applied  to 
the  crude  substance  in  its  normal  position  in  the  tissues,  the  colours 
obtained  are  always  dark  and  dirty-looking. 

1  Verhand.  d.  phi/s.-med.  Gesell.     Wiirzburg,  Bd.  xviir.  (1884),  Nr.  9,  S.  7. 

2  Zt.  f.  Biol.  Bd.  XXVI.  (1890),  S.  491. 

3  Virchow,  Compt.  Rend.  T.  xxxvii.  p.  492,  860. 

*  C.  Schmidt,  Ann.  d.  Chem.  u.  Pharm.  Bd.  ex.  (1859),  S.  250,  aud  Friedreich 
u.  Kekule',  Virchow's  Archiv,  Bd.  xvi.  (1859),  S.  50. 

5  Modrzejewski,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  i.  (1873),  S.  426. 

6  Weyl,  Zt.f.  phijsiol.'Chem.  Bd.  i.  (1877),  S.  339. 
"^  C.  Schmidt,  lor.  cit. 

8  Wien.  med.  Wochenschr.  No.  32,  S.  714. 

9  Compt.  Bend.  T.  Lxxx.  (1875),  p.  1288. 


CHEMICAL  BASIS   OF   THE   ANIMAL   BODY.         49 

Purified  lardacein  is  readily  soluble  in  moderately  dilute 
ammonia,  and  can,  by  evaporation,  be  obtained  from  this  solution 
in  the  form  of  tough,  gelatinous  flakes  and  lumps ;  in  this  form  it 
gives  feeble  reactions  only  with  iodine.  If  the  excess  of  ammo- 
nia is  expelled,  the  solution  becomes  neutral,  and  is  precipitated 
by  dilute  acids. 

Preparation.  The  gland  or  other  tissue  containing  this  body 
is  cut  up  into  small  pieces,  and  as  much  as  possible  of  the  sur- 
rounding tissue  removed.  The  pieces  are  then  extracted  several 
times  with  water  and  dilute  alcohol,  and  if  not  thus  rendered 
colourless  are  repeatedly  boiled  with  alcohol  containing  hydro- 
chloric acid.  The  residue  after  this  operation  is  digested  at  40° 
C,  with  active  artificial  gastric  juice  in  excess.  Everything  except 
lardacein,  and  small  quantities  of  mucin,  nuclein,  keratin,  together 
with  some  portion  of  the  elastic  tissue,  will  thus  be  dissolved  and 
removed.^  From  the  latter  impurities  it  may  be  separated  by 
fractional  decantation  of  the  finely-powdered  substance  from 
water,  alcohol,  and  ether. 

In  opposition  to  the  older  statements  it  has  recently  been 
stated  that  lardacein  may  be  digested  by  pepsin  in  presence  of 
hydrochloric  acid.^  The  writer's  own  experiments  lead  him  to 
believe  in  the  results  obtained  by  the  earlier  authorities. 


The  known  products  of  decomposition  of  proteids  are  very 
numerous,  varying  in  nature  and  relative  amount  with  the  con- 
ditions and  reagents  by  means  of  which  they  are  produced,  and 
it  may  be  similarly,  though  to  a  much  less  extent,  with  the  kind 
of  proteid  employed.  These  products  belong  for  the  most  part  to 
well-known  classes  of  chemical  substances,  and  in  many  cases 
representatives  of  several  consecutive  members  of  any  given 
homologous  series  are  obtained  during  the  decompositions. 

A  study  of  these  products  has  not,  however,  up  to  the  present 
time  thrown  any  extended  light  upon  the  more  minute  molecular 
structure  of  the  proteids,  and  the  reason  is  not  far  to  seek.  It 
consists  simply  in  the  fact  that  we  possess  no  guarantee  or  cri- 
terion of  the  purity  of  those  proteids  which  can  be  obtained  in 
sufficient  amounts  for  the  purposes  of  experiment.  They  may 
be,  and  probably  are,  mixtures  of,  it  may  be,  several  closely  allied 
substances,  so  that  the  numerous  products  which  arise  during  the 
decomposition  of  what  is  regarded  in  the  experiment  as  one 
uniform  substance,  represent  really  the  decomposition-products 
of  several  proteid  molecules,  and  thus  throw  no  light  on  the 
structure  of  any  one.     And  the  matter  is  still  further  complicated 

1  Kiiline  and  Rudneff,  Virchow's  Arch.  Bd.  xxxiii.  (1865),  S.  66. 

2  Kostjurin,  Wien.  med.  Jahrb.  1886,  S.  181. 

4 


50  PEOTEIDS. 

by  the  fact  that  the  final  products  of  any  given  decomposition  do 
not  at  all  necessarily  represent  the  primary  mode  of  breaking 
down  of  the  proteid  molecule ;  many  of  them  may  be  the  out- 
come of  some  secondary  decomposition  of  the  first-formed  pro- 
ducts. It  may  hence  suffice  to  give  a  short  account  of  the  more 
generally  important  researches  on  the  decompositions  of  proteids 
and  to  refer  the  reader  for  details  to  some  larger  work.^ 

The  products  of  the  decomposition  of  proteids  by  acids  (HCl) 
have  been  elaborately  studied  by  Hlasiwetz  and  Habermann.^ 
These  observers  subjected  proteids  (casein)  to  the  action  of  boil- 
ing concentrated  hydrochloric  acid  in  presence  of  stannous  chlo- 
ride for  three  days.  From  the  fluid  thus  obtained  they  were  able 
to  separate  out  by  repeated  crystallisations  leucin,  tyrosin,  glu- 
tamic and  aspartic  acids  and  ammonia ;  the  mother  liquor  from 
the  above  yielded  no  further  well-defined  substances.  Schutzen- 
berger,^  treating  proteids  in  presence  of  a  little  water  with  an 
excess  of  baryta  in  sealed  tubes  at  200  —  250°,  observed  a  more 
profound  breaking  down  of  these  substances  as  judged  by  the 
products  of  their  decomposition.  In  addition  to  the  products 
described  by  Hlasiwetz  and  Habermann  he  obtained  small  quan- 
tities of  carbonic,  oxalic,  and  acetic  acids,  together  with  other 
amido-acids  homologous  with  leucin,  amido-acids  of  other  series, 
leuceins,'*  gly co-protein,  tyroleucin,^  &c.  The  chief  difference  in 
the  results  obtained  by  the  two  sets  of  observers  turns  upon  the 
non-occurrence  of  carbonic,  oxalic,  and  acetic  acids  among  the 
products  of  the  action  of  hydrochloric  acid.  Drechsel  ^  has  how- 
ever shown  that  if  the  non-crystallisable  residue  from  Hlasiwetz 
and  Habermann's  experiments  be  appropriately  treated  with 
baryta  in  sealed  tubes  it  readily  yields  carbonic  acid,  so  that  the 
difference  may  turn  out  after  all  to  be  more  apparent  than  real. 
Interesting  as  are  the  above  researches  they  do  not  as  yet  enable 
us  to  form  any  clear  idea  of  the  probable  molecular  composition 
of  proteids.  According  to  Schiitzenberger  the  relative  amounts  of 
carbonic  acid  and  ammonia  which  make  their  appearance  are  the 
same  as  would  have  arisen  from  a  similar  treatment  of  urea  with 
caustic  baryta,  and  from  this  and  the  fact  of  the  preponderating 
appearance  of  amido-acids  by  the  action  of  the  alkaline  oxide, 

1  Ladenburg's  Handworterhuch  d.  Ckem.  Bd.  ill.  S.  541.  Beilstein's  Hdbch.  d. 
Chem.  Bd.  in.  8.  1258. 

2  Anzeia.  d.  Wien.  Akad.  1872,  S.  114;  1873,  Nr.  15.  Ann.  d.  Chem.  u.  Pharm. 
Bd.  159  (l'871),  S.  .304,  Bd.  169  (187.3),  S.  1.50.  Jn.  f.  prakt.  Chem.  (2)  Bd.  A'ir. 
S.  397.     See  also  E.  Schulze,  Zt.  f.  physiol.  Chem.  Bd.'ix.  (1885),  Sn.  63,  253. 

3  Ann.  de  Chim.  et  de  Phys.  (5  Se'r.)  T.  xvi.  (1879),  p.  289.  Bull,  de  la  Soc.  Chim. 
XXIII.  161,  193,  216,  242,  385,  4.33 ;  xxiv.  2,  145  ;  xxv.  147.  Also  in  Chern.  Centralb. 
1875,  Sn.  614,  631,  648,  681,  696;  1876,  S.  280;  1877,  S.  181.  Compt.  Rend.  T.  101, 
(1886),  p.  1267.  See  also  Nasse,  Pfliiger's  Arch.  Bde.  vi.  (1872),  589;  vii.  139; 
VIII.  381. 

*   Compf.  Bend.  T.  84  (1877),  p.  124. 

5  Ibid.  T.  106  (1888),  S.  1407. 

6  Jn.f  prakt.  Chem.  (N.  F.)  Bd.  xxxix.  (1889),  S.  425. 


CHEMICAL   BASIS   OF   THE   ANIMAL  BODY.  51 

he  regards  the  proteids  as  complex  ureides  :  that  is  to  say,  as 
combinations  of  urea  with  amido-acids  belonging  to  several  series 
such  as  the  leucic  and  aspartic.^  In  support  of  this  view  the 
work  of  Grimaux  '^  may  be  mentioned.  By  fusing  together  aspar- 
tic  anhydride  and  urea  he  obtained  a  substance  resembling  a 
proteid  in  several  of  its  reactions,  and  yielding  aspartic  acid, 
carbonic  acid  and  ammonia  by  treatment  with  baryta.  It  has 
not  however  as  yet  been  shown  that  this  substance  can  be  made 
to  yield  urea,  and  further,  no  one  has  ever  succeeded  in  obtain- 
ing urea  as  a  direct  product  of  the  decomposition  of  a  proteid. 
Further,  as  against  the  view  of  the  ureide  nature  of  proteids, 
Low's  views  as  to  the  probable  non-existence  of  amido-acid 
residues  in  the  proteid  molecule  must  not  be  lost  sight  of.^ 

The  older  statements  of  Bechamp  ^  and  Kitter^  as  to  the  formation 
of  urea  from  proteids  by  the  action  of  potassium  permanganate  are 
erroneous.^  The  most  recent  refutation  of  their  views  is  due  to 
Lossen/  who  finds  that  traces  of  guanidin  may  make  their  appear- 
ance hut  no  urea.  This  substance  might  however  be  easily  mistaken 
for  urea  since  its  compounds  with  oxalic  and  nitric  acids  closely 
resemble  those  of  urea  with  the  same  acids.  Although  guanidin 
when  boiled  with  sulphuric  acid  or  baryta  water  readily  yields  urea 
(and  simultaneously  ammonia)  this  can  in  no  way  be  taken  as  imply- 
ing a  possible  formation  of  urea  from  proteids  directly.  Quite 
recently  a  crystalline  base  called  '  lysatin, '  which  readil}^  yields  urea 
when  boiled  with  baryta  water, ^  has  been  isolated  from  among  the 
products  of  the  decomposition  of  casein  by  hydrochloric  acid  and 
chloride  of  zinc.  The  formula  of  this  base  is  given  as  CcHnNgO, 
thus  placing  it  in  close  compositional  relationship  with  kreatin 
C4H9N0O2  and  kreatinin  C4H7N3O. 

It  cannot  as  yet  be  said  that  we  possess  any  real  knowledge 
of  the  constitution  of  proteids,  and  the  question  will  probably 
remain  unsolved  until  some  entirely  new  departure  is  made  in 
attacking  the  problem,  or  until  some  new  property  of  proteids  is 
discovered  by  which  their  absolute  purity  may  be  determined  as 
the  necessary  preliminary  to  the  whole  investigation.  The  so- 
called  crystallised  proteids    (see  above,  p.  6)   have  not  as  yet 

1  For  Schiitzenberger's  most  recent  attempts  to  synthetise  proteids,  see  Coinpt. 
Rend.T.  112  (1891),  p.  198. 

2  Gaz.  med.  1879,  p.  521.     Compt.  Rend.  T.  93  (1881),  p.  771. 

3  Jn.  f.  prakt.  Chem.  Bd.  xxxi.  (1885),  S.  129.  „      ,  ^ 

4  Anil.  d.  Chem.  u.  Pharm.  Bd.  C.  (1856),  S.  247.  Compt.  Rend.  T.  i.xx.,  p.  866. 
T.  Lxxiii.,  p.  1.323. 

5  Ibid.  T.  LXXIII.,  p.  1219.  ^..  r,  ■  ,     ,^T    -r-  ; 

6  See  Stadeler,  Jn.f.  prakt.  Chem.  Bd.  i.xxii.  (1857),  S.  251.  Low,  Ibid.  (N.  i.) 
Bd.  III.  (1871),  S.  180.     Tappeiner,  Ber.  k.  Sachs.  Gesell.  1871. 

V  Ann.  d.  Chem.  u.  Pharm.  Bd.  201  (1880),  S.  369. 

8  Drechsel,  Ber.  d.  d.  Chem.  Gesell.  Jahrg.  xxiii.  (1890),  S.  3096.  Cf.  Siegfried, 
Ibid.  Jahrg.  xxiv.  S.  418. 


52  PEOTEIDS. 

been  prepared  in  sufficient  quantity  ^  to  admit  of  the  easy  and 
decisive  application  of  the  modern  metliods  of  organic  chemistry 
to  the  elucidation  of  their  molecular  structure.  Work  in  this 
direction  on  a  really  large  scale  could  scarcely  fail  to  yield  im- 
portant results.  Schrotter  ^  has  recently  described  the  preparation 
of  benzoylated  ethers  of  the  albumoses,  and  intends  to  apply  the 
method  to  other  proteids  and  to  study  the  products  of  decom- 
position and  oxidation  of  these  substances.  Whether  any  real 
advance  will  be  made  in  this  direction  cannot  be  foretold,  but 
this  new  departure  is  of  considerable  prospective  importance. 

No  account  of  the  constitution  of  proteids  would  be  complete 
without  a  reference  to  the  views  and  theories  of  Pfiliger,  and  of 
Low  and  Bokorny.  Pflliger  ^  starting  from  the  characteristic  dif- 
ferences between  the  products  obtained  by  decomposing  dead  pro- 
teids by  chemical  means  out  of  the  body,  and  the  products  which 
arise  by  the  natural  decomposition  (metabolism)  of  living  proteids 
(protoplasm)  in  the  body,  has  put  forward  a  view  as  to  the  dif- 
ference of  living  and  dead  proteid.  He  considers  that  in  dead 
proteid  the  nitrogen  exists  in  the  amide  form,  while  in  living 
proteid  it  is  present  in  the  less  stable  cyanic  form.  The  build- 
ing-up of  living  proteid  from  dead  he  regards  as  being  carried  on 
by  the  ether-like  union  of  the  isomeric  living  and  dead  proteid 
molecules,  accompanied  by  the '  elimination  of  water.  During  this 
process  the  nitrogen  of  the  dead  proteid  passes  into  the  cyanic 
condition,  and  if  this  is  repeated  and  accompanied  by  polymerisa- 
tion the  formation  of  a  large  and  unstable  living  proteid  molecule 
may  be  readily  accounted  for.  He  further  draws  attention  to  the 
readiness  with  which  polymerisation  occurs  in  the  cyanic  series 
and  the  extraordinarily  high  molecular  energy  of  cyanogen.  Low 
and  Bokorny  *  deal  also  with  the  probable  mode  by  which,  in  the 
case  at  least  of  plant  cells,  the  complex  proteid  molecule  may  be 
built  up  out  of  the  simpler  substances  from  which  these  obtain 
their  nitrogen.  They  consider  there  is  evidence  of  the  existence 
in  living  plant  cells  of  some  substance  of  an  aldehyde  nature. 
Starting  with  formic  aldehyde,  by  its  union  with  ammonia  the 
aldehyde  of  aspartic  acid  might  be  obtained,  and  by  polymerisa- 
tion of  the  latter  in  presence  of  sulphur  and  with  the  exit  of 
water  a  substance  with  the  same  composition  as  an  ordinary  proteid 
would  arise.  Their  speculations  are  ingenious,  but  it  cannot  by 
any  means  be  said  that  their  views  are  established.  Asparagin, 
from  which  aspartic  acid  is  readily  obtained,  undoubtedly  plays 
an  all-important   part  in  the  constructive  nitrogenous  metabol- 

1  But  see  Chittenden  and  Hartwell  Ji.  of  Physiol.  Vol.  xi.  (1890),  p.  435. 

2  Ber.  d.  deutsch.  chem.  Gesell  Jahrg.  xxii.  (1889),  S.  1950. 

3  Pfliiger's  Arch.  Bd.  x.  (1875),  S.  332. 

*  Low  and  Bokorny's  work  may  be  most  conveniently  quoted  by  reference  to  the 
following  volumes  of  Maly's  Jahresbe.richt  d.  Thierchem.  Bde.  x.  (1880),  S.  3  ;  xr.  391, 
394;  XII,  380;  xiii.  1;  xiv.  349,  474;  xvi.  8;  xvii.  (1887),  395.  See  also  Biol. 
Centralb.  Bd.  i.  (1881),  S.  193;  riii.  (1888),  S.  1. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  53 

ism  of  plants ;  but  as  yet  the  aldehyde  of  aspartic  acid  has  not 
been  prepared  by  any  chemical  means,  and  Baumann  ^  has  cast 
great  doubt  on  the  reliability  of  the  methods  by  which  the  above 
authors  have  endeavoured  to  prove  the  existence  of  aldehydes  in 
the  protoplasm  of  the  living  plant  cells.  And  it  is  probably  sig- 
nificant that  the  reactions  by  which  the  presence  of  the  aldehydes 
is  supposed  to  be  shown  are  only  well  marked  in  the  case  of  the 
cells  of  the  lowest  plants  ;  in  the  case  of  animal  cells  they  are 
more  usually  wanting. 

The  Enzymes  or  Soluble  Unorganized  Ferments.^ 

Chemists  have  for  a  long  time  been  familiar  with  an  extensive, 
and  still  increasing  class  of  reactions  which  occur  solely,  or  in 
some  cases  most  readily,  in  presence  of  minute  quantities  of  some 
substance  which  does  not  itself  appear  to  enter  directly  into  the 
reaction  ;  in  other  words  the  causative  agent  is  found  to  have 
itself  undergone  no  obvious  change  during  the  reactions  which  it 
has  set  up  between  the  other  substances.  Striking  instances  of 
such  reactions  are  observed  in  the  preparation  of  ether  from 
alcohol  by  means  of  sulphuric  acid  and  in  the  manufacture  of 
sulphuric  acid  itself.  In  the  former  case  a  small  quantity  of 
sulphuric  acid  is  theoretically  able  to  convert  an  indefinitely 
large  quantity  of  alcohol  into  ether,  and  in  practice  the  limit  is 
determined  simply  by  the  occurrence  of  secondary  decompositions 
between  the  reagents.  Similarly  during  the  manufacture  of  sul- 
phuric acid  a  minute  quantity  of  nitric  oxide  suffices  in  the  pres- 
ence of  water  to  convert  an  indefinitely  large  amount  of  sulphurous 
anhydride  into  sulphuric  acid.  Of  late  years  a  large  number  of 
reactions  have  been  found  to  depend  for  their  occurrence  upon  the 
presence  of  the  minutest  traces  of  water ;  thus  dry  chlorine  has 
no  action  on  dry  sodium,  and  dry  hydrochloric  acid  gas  and 
oxygen  do  not  react  even  when  exposed  to  bright  sunlight, 
neither  do  dry  oxygen  and  carbonic  oxide  explode  on  the  passage 
of  an  electric  spark.  The  fact'  of  immediate  interest  in  each  of 
the  above  instances  is  that  a  minute  trace  of  the  substance  which 
determines  the  occurrence  of  the  reaction  is  able  to  produce 
change  in  an  indefinitely  large  mass  of  the  other  reagents  without 
itself  undergoing  any  final  alteration.     Turning  to  the  chemistry 

1  Pfliiger's  Arch.  Bd.  xxix.  (1882),  S.  400.  See  also  Hoppe-Seyler,  Zt.  f.  physiol. 
Chem.  Bd.  x.  (1886),  S.  39. 

■^  It  appears  advisable  to  use  the  term  'enzyme'  (Kiihne,  Unters.  a.  d.  phi/siol. 
JhM.  Heidelh.  Bd.  i.  1878,  S.  293)  to  denote  the  soluble  unorganised  ferments  gen- 
erally, reserving  the  older  name  of  '  ferment '  for  the  organized  agents  such  as  yeast 
to  which  it  vv^as  first  applied.  If  this  be  done  it  will  be  convenient  to  use  the  expres- 
sion '  zymolysis  '  to  denote  the  changes  produced  by  the  enzymes  in  their  action  on 
other  substances,  and  to  apply  the  term  '  fermentation  '  to  the  action  of  the  organised 
ferments.  In  this  way  '  zymolysis '  corresponds  to  the  German  '  Ferment-wirkung,' 
and  'fermentation  '  to  'Gahrung.' 


54  ENZYMES   OR   SOLUBLE  FERMENTS. 

of  animal  and  vegetable  cells  it  is  found  that  in  many  cases  sub- 
stances may  be  extracted  from  them  which  possess  to  an  even 
more  striking  degree  the  property  of  inducing  change  in  an  indef- 
initely large  mass  of  certain  other  substances  without  themselves 
undergoing  any  observable  alteration.  These  agents  are  known 
as  the  enzymes  or  soluble  ferments,  and  the  essential  conception 
of  an  enzyme  is  summed  up  in  the  above  statement  of  the  most 
remarkable  characteristic  of  their  activity.  Further  investigation 
of  these  enzymes  shows  that  their  activity  is  dependent  upon 
many  subsidiary  factors  which  are  more  or  less  common  to  them 
all.  Thus  their  activity  is  largely  dependent  upon  temperature, 
being  absent  at  sufficiently  low  temperatures,  increasing  as  the 
temperature  is  raised  to  a  certain  optimal  point  which  varies 
slightly  for  different  enzymes,  then  again  diminisliing  as  the  tem- 
perature is  further  raised,  and  finally  disappearing.  By  the  action 
of  a  sufficiently  high  temperature  they  permanently  lose  their 
characteristic  powers  and  are  now  spoken  of  as  being  'killed.' 
Again  the  enzymes  are  extremely  sensitive  to  the  reaction, 
whether  acid,  alkaline,  or  neutral,  of  the  solutions  in  which  they 
are  working,  also  to  the  presence  or  absence  of  various  salts,  some 
of  which  merely  inhibit  their  action  while  others  permanently 
destroy  it ;  and  their  activity  is  in  all  cases  lessened  and  finally 
stopped  by  the  presence  of  an  excess  of  the  products  to  whose 
formation  they  have  given  rise.  It  has  been  already  said  that  an 
enzyme  may  be  killed  by  exposure  to  a  high  temperature,  but  this 
only  holds  good  when  they  are  in  solution,  or  if  in  the  solid  form 
they  are  heated  in  a  moist  condition.  When  perfectly  dry  they 
may  be  heated  to  100°  — 160°  without  any  permanent  loss  of 
their  powers.^  It  will  be  seen  that  so  far  the  enzymes  have  been 
characterised  solely  with  reference  to  the  peculiarity  of  their 
mode  of  action  and  to  the  influence  of  surrounding  conditions 
upon  that  activity,  and  the  question  of  their  probable  chemical 
composition  has  been  left  untouched.  Notwithstanding  the  fre- 
quent endeavours  which  have  been  made  to  prepare  the  enzymes 
in  a  pure  condition,  it  is  unwise  to  lay  any  great  stress  upon  the 
results  of  the  analysis  of  these  so-called  '  pure  ferments,'  bearing 
in  mind  that,  as  in  the  case  of  the  proteids,  no  criterion  of  their 
purity  exists.  This  much  however  may  be  said.  In  the  major- 
ity of  cases,  analysis  shows  that  their  composition  approximates 
more  nearly  to  that  of  a  proteid  than  of  any  other  class  of  sub- 
stances, and  this  is  apparently  true  even  when  they  do  not  yield 
to  any  marked  degree  the  reactions  (xanthoproteic,  &c.)  which 
are  characteristic  of  a  true  proteid.  Ordinarily  it  is  almost  im- 
possible to  obtain  an  enzyme  solution  of  any  considerable  activity 
which  is  free  from  proteid  reactions,  and  hence  many  authors  are 

1  Hiifner,  Jn.f.  prakt.  Chem.  Bd.  v.  (1872),  S.  372.  Al.  Schmidt,  Centralb.  f.  d. 
med.  Wiss.  1876,  "S.  510.  Salkowski,  Virchow's  ^?-c/i.  Bd.  i.xx.  (1876).  S.  158;  lxxxi. 
(1880),  S.  552.     Hiippe,  Miltheil.  d.  Kaiserl.  Gesundheitsamtes,  i.  1881. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  55 

inelined  to  regard  these  bodies  as  being  really  of  proteid  nature. 
But  this  is  a  point  which  is  as  yet  by  no  means  settled,  as  the  fol- 
lowing considerations  show.  The  sole  means  at  our  disposal  of 
determining  the  presence  of  an  enzyme  is  that  of  ascertaining  the 
change  which  it  is  able  to  bring  about  in  other  substances,  and 
since  the  activity  of  the  enzymes  is  extraordinarily  great,  a  minute 
trace  suffices  to  produce  a  most  marked  effect.  From  this  it  fol- 
lows that  the  purified  enzymes  which  give  distinct  proteid  reac- 
tions might  merely  consist  of  very  small  quantities  of  a  true 
non-proteid  enzyme  adherent  to  or  mixed  with  a  residue  of  inert 
proteid  material.  Again  on  the  other  hand  it  is  similarly  possible 
that  the  purified  enzymes  which  have  been  described  as  devoid  of 
proteid  reaction  really  consist  of  some  inert  non-proteid  material 
with  which  a  trace  of  what  is  really  a  true  proteid  enzyme  is  ad- 
mixed, the  amount  of  enzyme  being  too  small  to  yield  any  of  the 
reactions  characteristic  of  proteids.  The  occurrence  or  absence  of 
proteid  reactions  in  a  solution  of  an  enzyme  cannot  therefore  set- 
tle the  nature  of  the  enzyme,  and  for  similar  reasons  a  mere  anal- 
ysis of  the  separated  enzyme  is  also  inconclusive  ;  the  balance  of 
recent  opinion  appears  to  be  in  favour  of  the  view  that  the  enzymes 
are  proteid  in  nature,  but  this  is  still  an  open  question. 

Many  of  the  purified  enzymes  have  been  analj^zed  and  the  results 
show  in  many  cases  a  percentage  of  carbon  considerably  lower  than 
that  of  a  true  proteid.  Kiihne's  purest  trypsin  had  the  following 
percentage  composition:  C  =  47-22  — 48-09  :  H  =  7-15  — 7.44  ; 
N  =  12-59  — 13-41  ;  8  =  1-73  —  1-86.  For  other  analyses  see 
Aug.   Schmidt.^  Hufner/  Barth.^     But  see  also  Wurtz'^  and  Low.^ 

The  enzymes  are  possessed  of  certain  properties,  more  or  less 
common  to  them  all,  by  means  of  which  they  may  be  separated 
from  the  tissues  in  which  they  primarily  occur,  and  isolated  from 
the  solutions  thus  obtained.  Soluble  in  water,  they  may  be  pre- 
cipitated unchanged  from  this  solution  by  the  addition  of  an 
excess  of  absolute  alcohol.  They  may  also  in  many  cases  be 
precipitated  from  their  aqueous  or  other  solution  by  saturation 
with  neutral  ammonium  sulphate.^  They  are  conveniently  solu- 
ble in  glycerine  ''  from  which  they  may  as  before  be  precipitated 
by  an  excess  of  alcohol.  None  of  the  enzymes  are  diffusible  and 
hence  they  may  readily  be  freed  from  any  admixed  diffusible 

1  Inauq.  Diss.  Tubingen,  1871. 

2  Jn.  f.  prakt.  Cliem.  N.  F.  Bd.  v.  (1872),  S.  372. 

3  Ber.  d.  deutsch.  Chetn.  Gesell.  Jahrs.  xi.  (1878),  S.  474. 
*  Compt.  Rend.  T.  xc.  (1880),  p.  1379  ;  xci.  p.  787. 

5  Pfliiger's  Arch.  Bd.  xxvn.  (1882),  S.  203. 

6  Kiihne,  Verhand.  d.  naturh  -med.  Ver.  Heidelb.  in.  1886,  S.  463.  Also  Centralb. 
f.  d.  med.  Wiss.  1886,  Nr.  45.  Krawkow  (Russian).  See  Ber.  d.  deutsch.  chem.  Gesell. 
'Referatband.  1887,  S.  735  or  Malv's  .Jahre.sber.  xvn.  S.  466. 

'  V.  Wittich,  Pfluger's  Arch.  Bd.  ii.  (1869),  S.  193. 


56  ENZYMES   OR   SOLUBLE  EERMENTS. 

substances  by  means  of  dialysis.^  They  possess  further  the  re- 
markable property  of  adhering  with  great  tenacity  to  any  finely 
divided  precipitate  which  is  formed  in  the  solutions  in  which 
they  are  present,  more  particularly  if  the  precipitate  is  of  a  viscid 
or  gelatinous  nature.^  It  is  not  however  possible  to  base  upon 
the  above  properties  any  general  method  of  preparing  the  en- 
zymes which  is  equally  applicable  to  each  of  them;  some  are 
most  readily  prepared  in  a  fairly  pure  state  by  one  method,  some 
by  another,  and  very  many  by  the  conjoined  application  of  two 
methods.  A  further  consideration  must  not  be  lost  sight  of  in 
connection  with  the  separation  of  the  enzymes  from  the  parent 
tissues ;  this  is  the  fact  that  in  some  cases  the  enzymes  do  not 
exist  in  the  free  and  active  conditions  in  the  cells  of  the  respec- 
tive tissues,  but  in  the  form  of  an  inactive  antecedent,  to  which 
the  name  of  '  zymogen  '  is  usually  applied.^  Hence  to  obtain  an 
active  extract  it  is  frequently  necessary  to  treat  the  tissue  with 
some  such  reagent  as  shall  ensure  the  conversion  of  the  zymogen 
into  the  active  enzyme. 

During  prolonged  digestions  it  is  essential  to  insure  the  absence 
of  any  changes  due  to  the  development  of  bacteria  or  other  organ- 
isms. The  most  suitable  antiseptics  for  this  purpose  are  salicylic 
acid  (-1  p.c.)  and  thymol  (-5  p.  c).  These  reagents  are  dissolved 
in  a  small  quantity  of  alcohol  and  added  in  the  above  proportions 
to  the  digestive  mixture. 

It  is  frequently  a  matter  of  the  utmost  importance  to  determine 
whether  the  hydrolytic  power  of  any  given  preparation  is  due  to 
the  action  of  a  soluble  enzyme  or  of  a  ferment  (organised).  The 
discrimination  is  most  readily  effected  by  carrying  on  the  diges- 
tion in  presence  of  chloroform,  which  is  inert  towards  the  enzymes 
but  inhibits  the  activity  of  ferment  organisms.* 

Special  DESCRiPTioisr  of  the  more  important  Enzymes.^ 
Ptyalin. 

While  occurring  chiefly  and  characteristically  in  saliva,  a  similar 
enzyme  may  be  obtained  in  minute  amount,  but  fairly  constantly, 
from  almost  any  tissue  or  fluid  of  the  body,  more  particularly  in 
the  case  of  the  pig.  It  was  first  separated  out  from  saliva,  but 
in  an  impure  condition,  by  Mialhe,  who  precipitated  the  saliva 
with  an  excess  of  absolute  alcohol.''     It  has  been  prepared  in  the 

1  Maly,  Pfliiger's  Arch.  Bd.  ix.  (1874),  S.  592. 

-  Briicke,  Sitzb.  d.  Wien.  Akad.  Bd.  xliii.  (1861),  S.  601.  Danilewsky,  Vir- 
chow's  Arch.  Bd.  xxv.  (1862),  S.  279.  Cohnheim,  Virchow's  Arch.  Bd.  xxviii. 
(1863),  S.  241. 

3  Heidenhain,  Pfliiger's  Arch.  Bd,  x.  (1875),  S.  583. 

*  Miintz,  Compt.  Rend.  T.  lxxx.  (1875),  p.  1255. 

5  Consult  the  article  '  Fermente'  by  Emmerling  iu  Ladenburg's  HandwOrterbuck 
d,  Chem.  Bd.  iv.  1887,  S.  95. 

6  Compt.  Rend.  T.  xx.  (1845),  pp.  954,  1485. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  57 

purest  (?)  form  by  Cohnheim.^  His  method  consists  in  the  addi- 
tion of  phosphoric  acid  to  the  saliva  until  it  is  strongly  acid ;  the 
mixture  is  then  neutralised  by  the  careful  addition  of  lime-water, 
whereupon  a  copious  precipitate  of  phosphate  of  lime  is  formed. 
This  carries  down  with  it  a  large  proportion  of  the  proteids  which 
are  present,  together  with  all  the  ptyalin.  On  extraction  of  the 
precipitate  with  a  volume  of  water  equal  to  that  of  the  saliva 
originally  employed,  the  enzyme  passes  chiefly  into  solution,  since 
it  is  less  firmly  adherent  to  the  precipitate  than  are  the  pro- 
teids ;  it  may  now  be  purified  still  further  by  repeating  the  above 
process  and  finally  precipitating  with  absolute  alcohol.  Prepared 
in  this  way,  the  enzyme  is  obtained  as  a  fine  white  amorphous 
powder.  Dissolved  in  water  it  is  extremely  active  in  hydrolysing 
starch,  and  the  solution  yields  none  of  the  reactions  most  typically 
characteristic  of  proteids.  On  these  grounds  it  is  asserted  that 
ptyalin  is  not  a  proteid,  but  the  evidence  is  not  conclusive. 
More  recently  this  enzyme  has  been  prepared  as  follows.^  Saliva 
is  diluted  with  an  equal  volume  of  water,  and  saturated  with 
neutral  ammonium  sulphate.  The  precipitate  thus  formed  is 
treated  on  the  filter  for  five  minutes  with  strong  alcohol,  removed 
from  the  filter,  and  further  treated  with  absolute  alcohol  for  one 
or  two  days.  It  is  now  dried  at  30°,  and  yields,  on  extraction 
with  a  volume  of  water  equal  to  that  of  the  original  saliva,  a 
solution  which  is  actively  zymolytic,  and  is  stated  to  be  free  from 
all  proteid  reactions.  The  hydrolytic  activity  of  ptyalin  is  most 
marked  in  neutral  or  nearly  neutral  solutions.^ 

An  amylolytic  enzyme  is  found  in  urine."* 

No  experiments  have  as  yet  established  the  existence  of  any 
zymogen  of  ptyalin  (ptyalinogen).^ 

The  amylolytic  enzyme  of  the  pancreas. 

The  secretion  of  the  pancreas  is  even  more  active  than  saliva 
in  effecting  the  hydrolysis  of  starch.^  This  property  is  dependent 
upon  the  presence  in  this  secretion  of  an  enzyme  which  in  many 
ways  closely  resembles  ptyalin,  but  differs  from  it  markedly  in 
its  greater  power  of  effecting  a  more  complete  decomposition  of 
the  starch  than  can  ptyalin.  Under  ordinary  conditions  the  only 
sugar  formed  by  the  action  of  ptyalin  on  starch  is  maltose  ;  if, 
however,  the  action  is  prolonged,  small  amounts  of  dextrose  may, 
it  is  stated,  also  make  their  appearance  as  the  result  of  the  fur- 

1  Virchow's  Arch.  Bd.  xxviii.  (1863),  S.  241. 
^  Krawkow,  loc.  cit. 

3  Langley  and  Eves,  Jl.  of  Physiol.  Vol.  iv.  (1882),  p.  18. 
*  For  litt.  see  ref.  1,  sub  Pepsin,  p.  61. 
5  Langley,  JL  of  Physiol.  Vol.  in.  (1881),  p.  288. 

''  Kiihne,  Lehrh.  d.  phijsiol.  Chem.  1868,  S.  117.  Maly  in  Hermann's  Hdbch.  d. 
Physiol.  Bd.  v.  2,  S.  194. 


58  ENZYMES   OR   SOLUBLE  FERMENTS. 

ther  action  of  the  enzyme  on  the  first-formed  maltose.^  But  this 
is  by  no  means  quite  certainly  the  case,  and  without  doubt  no 
dextrose  is  obtained  during  a  digestion  of  moderate  duration. 
The  pancreatic  enzyme,  on  the  other  hand,  not  only  rapidly  con- 
verts starch  into  maltose,  but  further  converts  this  maltose  into 
dextrose  in  considerable  quantity  during  a  digestion  of  relatively 
short  duration  in  comparison  with  that  required  for  its  production 
by  the  action  of  ptyalin.^  The  secretion  of  the  pancreas  is  of  ex- 
tremely complicated  composition,  and  contains  in  addition  to  the 
amylolytic  at  least  two  other  well  characterised  enzymes ;  from 
these  the  former  has  as  yet  been  only  very  imperfectly  separated, 
so  that  scarcely  anything  is  known  of  its  chemical  nature  as  dis- 
tinct from  its  converting  powers.  According  to  von  Wittich  the 
amylolytic  enzyme  is  separable  from  the  others  by  treating  the 
gland  with  ether  and  alcohol  before  its  extraction  with  glycerine, 
to  which  reagent  it  then  yields  only  the  amylolytic  enzyme ;  ^ 
Hiifner,  however,  obtained  a  mixture  of  enzymes  by  von  Wittich's 
method.*  Experiments  on  the  separation  of  the  enzymes  have 
also  been  made  by  Danilewsky  ^  and  Paschutin ;  ^  but  the  most 
successful  outcome  of  any  method  which  may  be  employed  simply 
results  in  the  production  of  an  extract  which  is  preponderatingly 
amylolytic,  but  is  by  no  means  free  from  the  other  enzymes.  An 
active  amylolytic  extract  is  best  prepared  by  Roberts'  method,'''  in 
which  the  finely  minced  pancreas  is  extracted  for  five  or  six  days 
with  four  times  its  weight  of  25  p.c.  alcohol,  the  mixture  being 
frequently  stirred.  The  pancreas  of  the  pig  yields  the  most  cer- 
tainly active  extracts,  and  more  particularly  if  the  gland  is  kept 
for  24  hours  after  removal  from  the  body,  and  is  then  treated  for 
a  few  hours  with  dilute  ('5  p.c.)  acetic  acid  before  its  final  ex- 
traction with  alcohol. 

Benger's  'liquor  paiicreaticus '  is,  when  freshly  prepared,  possessed 
of  extraordinarily  active  amylolytic  powers.  Erom  it  an  extremely 
pure  and  active  solution  of  the  enzyme  may  be  obtained  by  adding  to 
it  four  times  its  volume  of  strong  alcohol  and  filtering  off  the  precipi- 
tate thus  formed ;  the  precipitate  is  then  rapidly  washed  with  alcohol, 
dried  in  the  air,  and  dissolved  in  water. 

The  secretion  and  extracts  of  the  small  intestine  possess  to  a 

1  Musculus  uud  Gruber,  Zt.  f.  physiol.  Chem.  Bd.  ii.  (1878),  S.  177.  Musculus 
uud  V.  Meriug,  Ibid.  S.  403.     v.'Mering,  Ibid.  Bd.  v.  (1881),  S.  185. 

^  Brown  and  Heron,  Liebig's  Ann.  Bd.  cxcix.  (1879),  S.  16.5.  Ibid.  Bd.  cciv. 
(1880),  S.  228.  Proc.  Roi/.  Soc.  No.  204  (1880),  p.  393.  Confirmed  also  by  the 
author's  own  experiments. 

3  Pfliiger's  Arch.  Bd.  ii.  (1869),  S    198. 

*  Hiifner,  Jn.f.  prakt.  Chem.  N.  F.  Bd.  v.  (1872),  S.  372. 

5  Virchow's  Arch.  Bd.  xxv.  (1862),  S.  279.  But  see  Lossnitzer,  Diss.  Leipzig, 
1864. 

«  Arch./.  Anat.  u.  Physiol.  Jahrg.  1873,  S.  382. 

7  Proc.'Roy.  Soc.  Vol.  xxxii.  (1881),  p.  145.  See  also  Digestion  and  Diet,  1891, 
pp.  16,  69. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.  59 

slight  extent  the  power  of  slowly  hydrolysing  starch  into  maltose  • 
the  conversion  being  more  rapid  if  portions  of  the  mucous  mem- 
brane of  the  intestine  be  finely  divided  and  immersed  in  the 
starch  solution.^  The  tissue  and  its  extracts,  on  the  other  hand, 
possess  to  a  very  marked  extent  the  power  of  rapidly  effecting  a 
conversion  of  maltose  into  dextrose  ;  this  is  of  great  physiological 
significance,  inasmuch  as  it  points  to  the  probability  that  the  car- 
bohydrates are  absorbed  from  the  intestine  as  dextrose  and  not  as 
maltose,  —  a  view  which  is  supported  by  the  fact  that  maltose  does 
not  appear  to  be  capable  of  direct  assimilation,  but  is  excreted 
largely  unchanged  if  injected  into  the  blood.^  If  this  be  so,  then 
it  is  as  dextrose  that  the  liver  receives  its  supply  of  carbohydrate 
material  for  the  formation  of  glycogen,  —  a  fact  which  is  of  no 
small  interest  when  we  know  that  the  liver  discharges  the  carbo- 
hydrate which  results  from  the  reconversion  of  glycogen  into 
sugar  as  dextrose.^     (See  also  sub  glycogen.) 

Cane-sugar  has  been  shown  b^'  Bernard  to  be  similarly  incapable  of 
assimilation;  if  injected  into  the  blood  it  is  excreted  in  the  urine 
unchanged.  When  taken  through  the  alimentary  canal  it  is  probably 
inverted  or  converted  into  a  mixture  of  dextrose  and  Isevulose,  which 
are  then  assimilable. 

The  conversion  of  hepatic  gh'cogen  into  sugar  as  a  preliminary  to  its 
discharge  from  the  liver  has  more  usually"  been  regarded  as  dependent 
upon  the  activity  of  some  special  hejiatic  enzyme.  This  view  is  now 
no  longer  tenable  in  face  of  the  negative  evidence  as  to  its  existence 
obtained  by  more  recent  observers.^     (See  also  sub  glycogen.) 

Pepsin. 

This  is  the  characteristic  proteolytic  enzyme  of  gastric  juice. 
It  was  first  separated  out  in  an  approximately  pure  form  by 
Brlicke.^ 

His  method  was  as  follo^vs.  The  mucous  membrane  of  the  stomach 
is  separated  from  the  muscular  coats,  finely  chopped  and  digested  with 
a  large  volume  of  5  p.  c.  phosphoric  acid.  The  fluid  thus  obtained  is 
strained  off  through  linen,  and  filtered,  and  lime-water  is  added  until 
the  reaction  is  just  not  quite  neutral;  by  this  means  a  precipitate  of 

1  Brown  and  Heron,  Proc  Hoy.  Soc.  No.  204  (1880),  p.  393.  Liebig's  Ann.  Bd. 
CCiv.  (1880),  S.  228.  Vella,  Moleschott's  Untersuch.  zu  Nutuiiehre,  Bd.  xiii.  (1881), 
S.  40.     Bourquelot,  Coiapt.  Rend.  T.  xcvii.  (1883),  p.  1000. 

2  Bimmermann,  Pfliiger's  Arch.  Bd.  xx.  (1879),  S.  201.  Philips  (Dutch  Diss.). 
See  Maly's  Bericht,  Bd.  xi.  (1881),  S.  60.  Dastre  et  Bourquelot,  Compt.  Rend.  T. 
xcviii.  (1884),  p.  1604.  Bourquelot,  Jn.  de  I'Anat.  et.  de  la  Physiol.  T.  xxii.  (1886), 
p.  161. 

3  Nasse,  Pfluger's  Arch.  Bd.  xiv.  (1877),  S.  479.  Seegen,  Ibid.  xix.  (1879),  S. 
123.  Seegen  und  Kratschmer,  Ihid.  xxii.  (1880),  S.  206.  Kiilz,  Ibid.  xxiv.  S.  52. 
Musculus  und  v.  Mering,  Zt.  f.  phijsiol.  Chem.  Bd.  ii.  (1878),  S.  417. 

-*  Eves,  Jl.  of  Phi/siol.  Vol.  v.  (1884),  p.  342.  (Gives  litt.  to  date.)  Dastre, 
Arch,  de  Physiol.  (4)'  T.  i.   (1888),  p.  69. 

5  Sitzb.  d.  Wien.  Akad.  Bd.  XLiii.  (1861),  S.  601.  See  also  his  Varies,  iiber 
Physiol,  (sub  pepsin). 


60  ENZYMES   OR   SOLUBLE  FERMENTS. 

calcium  phosphate  is  obtained  to  wliich  all  the  pepsin  is  adherent. 
The  precipitate  is  now  filtered  off,  dissolved  in  a  minimal  amount  of 
dilute  hydrocliloric  acid  and  again  precipitated  by  the  addition  of  lime- 
water;  this  second  precipitation  frees  the  pepsin  largely  from  the 
proteids  which  were  at  first  carried  down  with  it.  This  second  pre- 
cipitate is  now  as  before  dissolved  in  dilute  hydrochloric  acid.  From 
this  the  pepsin  is  separated  as  follows.  Cholesterin  is  dissolved  in  a 
mixture  of  four  parts  of  alcohol  and  one  of  ether,  and  this  solution  is 
introduced  below  the  solution  of  pepsin  by  means  of  a  long  thistle- 
tube.  As  soon  as  the  cholesterin  comes  in  contact  with  the  water  it 
separates  out  and  the  separation  is  completed,  as  a  finely  granular 
mass,  by  violently  shaking  the  vessel  in  which  the  mixture  is  con- 
tained. The  pepsin  adheres  now  to  the  cholesterin,  which  is  filtered 
off,  washed  first  with  water  faintly  acidulated  with  acetic  acid  and 
finally  with  pure  water.  On  treating  the  mass  with  pure  ether  in  a 
separating-funnel  the  cholesterin  goes  into  solution  in  the  ether  which 
forms  an  upper  layer,  below  which  is  an  aqueous  solution  of  pepsin, 
which  must  be  shaken  up  several  times  with  renewed  portions  of  ether 
until  all  the  cholesterin  has  been  extracted.  The  aqueous  solution  of 
the  enzj'me  thus  obtained  is  exposed  to  the  air  until  it  is  free  from 
ether,  and  is  then  filtered.  It  may  be  further  purified  by  dialysis, 
and  is  now  found  to  give  none  of  the  reactions  characteristic  of  pro- 
teids, and  to  be  precipitable  only  by  the  acetates  of  lead.  It  yielded 
no  trace  of  opalescence  on  the  addition  of  tannic  acid,  though  this  is 
capable  of  detecting  one  part  of  proteid  in  100,000  of  solvent.'^ 

From  the  reactions  of  the  pepsin  solution  obtained  by  Briicke's 
method,  it  seems  justifiable  to  consider  that  the  enzyme  is  not 
really  a  proteid.  The  same  conclusion  may  be  deduced  from  the 
more  recent  investigation  of  Sundberg.^  No  analyses  of  purified 
pepsin  appear  to  have  been  made  as  yet,  so  that  the  views  as  to 
its  non-proteid  nature  are  based  solely  upon  the  reactions  of  its 
solutions  as  described  by  Briicke  and  Sundberg,  reactions  which, 
as  already  pointed  out,  are  not  really  conclusive. 

Preparation  of  peptic  digestive  fluids.  If  a  few  drops  of  a 
glycerine  extract  of  gastric  mucous  membrane  be  added  to  dilute 
(•2  p.  c.)  hydrochloric  acid,  or  if  the  tissue  be  simply  extracted  for 
a  short  time  with  the  dilute  acid  and  the  extract  be  filtered,  a 
solution  is  obtained  which  suffices  for  demonstration  and  ordi- 
nary purposes.^  When  however  a  peptic  extract  is  required  for 
research  purposes  it  is  essential  to  adopt  some  more  elaborate 
method  which  yields  a  product  as  free  as  possible  from  admixed 
substances ;  one  of  the  best  is  that  of  Maly.*  The  mucous  mem- 
brane is  digested,  as  in  Briicke's  method,  with  phosphoric  acid 
and  the   fluid  precipitated  with  lime-water.     The  precipitate  of 

1  Hofmeister,  Zt.  f.  physiol.  Chem.  Bd.  ir.  (1878),  S.  292. 

2  Zt.  f.  physiol.  Chem'.'QiX.  ix.  (1883),  S.  319.  But  see  Low,  Pfliiger's  Arch.  Bd. 
XXXVI.  (1885),  S.  170. 

3  See  also  Kiiline  and  Chittenden,  Zt.  f.  Biol.  Bd.  xix.  (1883),  S.  184. 

4  Pfliiger's   Arch.  Bd.  ix.  (1874),  S.  592. 


CHEMICAL   BASIS   OF   THE  ANIMAL   BODY.  61 

calcium  phosphate  is  then  filtered  off,  washed,  and  dissolved  in 
dilute  hydrochloric  acid,  and  this  solution  is  then  dialysed  until 
it  is  free  from  chlorine  and  phosphates,  and  on  acidulating  with 
hydrochloric  acid  is  ready  for  use. 

Owing  to  the  relatively  slow  diffusibility  of  albumoses  and  peptones, 
mere  dialysis  of  a  solution  of  pepsin  in  which  these  substances  are 
present  does  not,  within  any  reasonable  time,  suffice  to  yield  an  even 
comparatively  pure  solution  of  the  enzyme. 

Many  forms  of  commercially  prepared  pepsin  are  obtained  by  digest- 
ing tlie  gastric  mucous  membrane  with  dilute  hydrochloric  acid;  the 
solution  thus  obtained  is  tlien  saturated  with  some  salt  such  as  NaCl, 
MgS04  or  CaClo,  whereupon  a  scum  rises  to  the  surface,  consisting 
chiefly  of  proteid  matter  to  which  the  pepsin  is  adherent.  This  scum 
is  then  removed,  frequently  mixed  with  milk-sugar  and  dried  at  a  low 
temperature.^ 

Pepsin  does  not  exist  preformed  in  the  cells  of  the  gastric 
glands,  but  as  a  zymogen  to  which  the  name  of  pepsinogen  is 
given ;  this  is  readily  converted  into  pepsin  by  the  action  of 
hydrochloric  acid.^ 

Unlike  ptyalin  the  hydrolytic  activity  of  pepsin  is  manifested 
only  in  presence  of  an  acid.  The  most  efficient  acid  in  this 
respect  for  artificial  digestions  is  hydrochloric  of  a  strength  of 
•2  p.  c.^  The  average  percentage  of  this  acid  may  be  stated  as 
■2  p.  c.  in  normal  gastric  juice,  but  it  varies  slightly  in  the  case 
of  different  animals.^  Other  acids  may  be  substituted  for  the 
hydrochloric,  the  optimal  percentage  varying  for  the  several 
acids.^ 

A  remarkable  peptonising  enzyme  (papain),  exits  in  the  milky  juice 
of  an  East  and  West  Indian  plant,  Carica  Papaya.  Any  description 
of  this  enzyme  and  its  properties  lies  outside  the  scope  of  this  work; 
all  necessary  information  may  be  obtained  by  referring  to  the  papers 
quoted  below.® 

Traces  of  pepsin  and  other  enzymes  are  frequently  found  in 
urine  ;  the  literature  of  the  subject  up  to  the  present  date  is  fully 
quoted  in  the  papers  to  which  a  reference  is  here  given.*" 

1  Scheffer.  See  abstract  in  Maly's  Jahresbericht.  Tid.  iii.  (1873),  8.  159.  Sellde'u 
(Swedish),  Ibid.  S.  159. 

-  Ebstein  und  Griitzner,  Pfliiger's  Arcli.  Bd.  viii.  (1874),  S.  122.  Langlev,  Jl.  of 
Physiol.  Vol.  III.  (1881),  p.  278.  Langlev  and  Edkins,  Ibid.  Vol.  xn.  (1886)"  p.  37i. 
Podwvssozkv,  Pfliiger's  Arch.  Bd.  xxxix."  (1886),  S.  62. 

3  Ad.  Maver,   Zt.f.  Biol.  Bd.  xvii.  (1881),  S.  356. 

*  Bidder  und  Schmidt,  Die  Verdauimgssafte,  Leipzig,  1852,  S.  46.  Heidenhaiu, 
Pfliiger's  Arch.  Bd.  xix.  (1879),  S.  152. 

5  David.sonund  Dieterich,  Arch.  f.  Anat.  u.  Physiol.  Jahrg.  1860,  S.  688.  Petit. 
See  ref.  in  Maly's  Bericht.  Bd.  x.  1880,  S.  308.     Also  Ad.  Mayer,  he.  cit. 

«  Wurtz  et  Bouchut,  Compt.  Rend.  T.  lxxxix.  (1879),  p.  425.  Wurtz,  Ibid.  T. 
xc.  p.  1379;  T.  xci.  p.  787.  Polak  (Dutch).  See  Abst.  in  Malv's  Jahresber.  1882, 
S.  254.     Martin,  Jl.  of  Physiol.  Vol.  v.  (1883),  p.  213 ;  vi.  p.  336.' 

'  Stadelmann,  Zl.  f.  Biol.  Bd.  xxiv.  (1888),  S.  226.  See  also  Wasilewski 
(Russian).  Abst.  in  Maly's  Bericht.  (1887),  S.  193.  H.  Hoffmann,  Pfliiger's  Arch. 
Bd.  XLi.  (1887),  S.  148.     Helwes,  Ibid.  Bd.  xliii.  (1888),  S.  384. 


62  ENZYMES   OR   SOLUBLE  FERMENTS. 

Trypsin. 

The  proteolytic  enzyme  of  pancreatic  juice.  This  appears  to 
have  been  first  separated  from  the  other  enzymes  which  exist 
in  pancreatic  juice  by  Danilewsky.^  More  recently  Ktihne  has 
prepared  it  in  quantity  and  in  what  must  be  presumed  to  be  a 
pure  (?)  form,  by  an  elaborate  and  lengthy  process,  for  the  details 
of  which  his  original  work  must  be  consulted.^  The  composition 
of  the  enzyme  as  prepared  by  Ktihne  was  found  to  be  remarkably 
complex,  as  shown  by  the  fact  that  when  dissolved  in  water  and 
boiled  it  is  split  up  with  the  formation  of  20  p.  c.  coagulated 
proteid  and  80  p.  c.  albumose.  It  might  at  first  sight  appear 
probable  from  this  that  the  purified  enzyme  was  in  reality  a 
mixture  of  the  true  enzyme  with  other  substances  (proteid)  to 
whose  decomposition  on  boiling  the  coagulated  proteid  and  albu- 
mose were  due,  and  some  authors  have  taken  this  view/^  This 
seems  however  to  be  negatived  by  the  fact  that  Kuhne  digested 
his  trypsin  for  several  weeks  in  dilute  alkaline  solution  and  did 
not  observe  the  formation  of  the  least  trace  of  peptone,  leucin, 
or  tyrosin.  The  percentage  composition  of  the  enzyme  has  been 
quoted  on  p.  55,  from  which  it  appears  to  contain  distinctly  less 
carbon  than  a  true  proteid. 

Preparation  of  solutions  of  trypsin  for  digestion  experiments. 
The  following  method  due  to  Kiihne  yields  an  extraordinarily 
pure  and  active  tryptic  solution  ;  unfortunately  it  is  a  somewhat 
lengthy  process.* 

One  part  by  weight  of  pancrea.s  which  has  been  extracted  with 
alcohol  and  ether  is  digested  at  40°  for  4  hours  with  5  parts  of  -1  p.  c. 
salicylic  acid.  The  residue  after  being  squeezed  out  is  further 
digested  for  12  hours  with  5  parts  of  "25  p.  c.  Na2C03,  and  the  residue 
is  again  squeezed  out.  The  acid  and  alkaline  extracts  are  now  mixed 
together,  the  whole  made  up  to  -25  —  -5  p.  c.  NajCOs,  and  digested 
for  at  least  a  week  in  presence  of  '5  p.  c.  thymol.  By  this  means  all 
the  first  formed  albumoses  are  fully  converted  into  peptones;  this  is 
essential.  At  the  end  of  the  week  the  fluid  is  allowed  to  stand  in 
the  cold  for  24  hours,  filtered,  faintlj^  acidulated  with  acetic  acid,  and 
saturated  with  neutral  ammonium  sulphate.  By  this  means  all  the 
trypsin  is  separated  out  and  may  be  collected  on  a  filter,  where  it  is 
washed  with  the  ammonium  salt  (sat.  sol.)  till  free  from  peptones. 
It  is  now  finally  dissolved  off  the  filter  in  a  little  -25  p.  c.  solution 
of  NagCOs,  to  which  thymol  is  added  and  thus  an  extremely  active 
and  very  pure  digestive  solution  is  obtained.  Ten  grams  of  the 
original  pancreas  yield  80  — 100  c.  c.   of  extract. 

1  Virchow's  Arch.  Bd.  xxv.  (1862),  S.  279. 

^   VerhandL  d.  naturlnst.-med.  Ver.  Heidelbg.  (N.F.),  Bd.  i.  (1876),  S.  194. 
3  Low,  Pfliiger's  Arch.  Bd.  xxvii.  (1882),  S.  209. 

*  Verhand.  d.  naturhist.-med.  Ver.  Heidelbg.  (N.F.),  Bd.  in.  (1886),  S.  463.  Also 
Centralb.f.  d.  med.  Wi&s.  1886,  Nr.  4,5. 


CHEMICAL   BASIS   OF   THE   ANIMAL   BODY.  63 

Although  Benger's  liquor  pancreaticus '  contains  in  addition  to 
the  enzymes  both  leucin  and  tyrosin  together  with  proteids,  it  is  so 
actively  proteolytic  that  the  small  amount  required  to  yield  an  active 
digestive  solution  introduces  an  amount  of  impurities  which  may  he 
neglected  in  many  cases.  The  above  impurities  may  be  largely  got 
rid  of  by  precipitating  out  the  enzymes  with  alcohol  as  described  on 
p.  58. 

Although  trypsin  exhibits  its  liydrolytic  powers  to  the  greatest 
advantage  in  presence  of  an  alkali,  its  activity  is  scarcely  so 
directly  related  to  the  alkali  as  is  that  of  pepsin  to  dilute  hydro- 
chloric acid.  Thus  it  will  digest  proteids,  although  much  more 
slowly  in  a  neutral  solution  and  even  in  presence  of  dilute  (•012 
p.  c.)  hydrochloric  acid,  but  the  slightest  excess  (•!  p.  c.)  of  the 
acid  destroys  it.^  In  connection  with  these  statements  it  must 
however  be  borne  in  mind  that  proteids  have  the  power  of  readily 
combining  with  acids,  hence  the  addition  of  say  '1  p.  c.  of  hydro- 
chloric acid  to  a  digestive  mixture  does  not  imply  that  there  is 
then  -1  p.  c.  of  free  acid  in  the  solution.^ 

This  comparative  independence  of  tryptic  activity  in  its  rela- 
tions to  the  reaction  of  the  digestive  mixture  is  doulDtless  of  con- 
siderable physiological  significance.  The  reaction  of  the  contents 
of  the  small  intestine  is  very  variable.  The  chyme  as  discharged 
from  the  stomach  is  of  course  acid,  and  this  acidity  is  largely 
diminished  by  the  advent  of  the  strongly  alkaline  bile  and  pan- 
creatic juice,  so  that  the  reaction  may  become  alkaline  within  a 
short  distance  of  the  pylorus.  On  the  other  hand  the  alkaline 
reaction  may  not  be  at  all  appreciable  until  the  lower  end  of  the 
intestine  is  reached,  and  frequently,  at  least  in  dogs,  the  reaction 
is  faintly  acid  throughout,  whether  they  are  fed  on  proteids  or  on 
a  mixture  of  carbohydrates  and  fat.^  The  acidity  in  the  latter 
case  is  not  surprising  bearing  in  mind  the  readiness  with  which 
the  carbohydrates  undergo  a  lactic  fermentation,  especially  inside 
the  intestine,  and  it  might  therefore  have  been  abnormal  in  the 
dog  whose  food  does  not  normally  contain  carbohydrates.  On 
the  other  hand  in  man,  living  on  a  mixed  diet,  the  possibility  of 
a  lactic  fermentation  is  always  present.*  It  is  impossible  to  make 
any  general  statement  as  to  the  reaction  of  the  contents  of  the 
small  intestine  ;  it  varies  at  different  times,  and  depends  upon  the 

1  Kiihne,  Virchow's  Arcli.  Bd.  xxxix.  (1867),  S.  130.  Heideuhain,  Pfliiger's 
Arch.  Bd.  x.  (1875),  S.  570.  May.s,  Untersuch.  a.  d.  physiol.  Inst.  Heidelb.  Bd.  iii. 
(1880),  S.  378.  Lindberger  (Swedish).  See  Abst.  in  Maly's  Jahresher.  Bd.  xiii. 
(1883),  S.  280. 

2  Szabd,  Zt.f.  physiol.  Chein.  Bd.  i.  (1877),  S.  140.  Danilewsky,  Centralb.  f.  d. 
med.  Wiss.  1880,  No.  51.  v.  d.  Velden,  Beutsch.  Arch.  f.  Klin.  Med.  Bd.  xxvii. 
(1880),  S.  186.     Cf.  Langley  and  Eves,  Jl.  of  Physiol.  Vol.'iv.  (1882),  p.  19. 

3  Schmidt-Mlilheim,  Arch.  f.  Physiol.  Jahrg.  1879,  S.  39.  Cash,  Ibid.  1880,  S. 
323.     Lea,  Jl.  Physiol.  Vol.  xi.  (1890),  p.  256. 

*  According  to  Hammarsten  the  gastric  mucus  contains  an  enzyme  which 
converts  lactose  (milk-sugar)  into  lactic  acid.  See  Maly's  Bericht.  Bd.  ii.  (1872),  S 
124. 


64  ENZYMES   OR   SOLUBLE  FERMENTS. 

kind  and  relative  aiuount  of  the  several  food-stuffs,  the  changes 
these  undergo  and  the  amount  of  alkaline  secretions  with  which 
they  are  mixed.  All  the  evidence  we  do  possess  leads  to  the 
belief  that  intestinal  digestion  to  be  of  use  must  be  capable  of 
being  carried  on  in  a  mixture  which  may  be  alkaline,  or  neutral, 
or  even  frequently  acid.  Although  the  acidity  of  the  intestinal 
contents  may  be  due  to  hydrochloric  acid  in  the  upper  end  of  the 
duodenum,  the  acidity  is  elsewhere  much  more  probably  due  to 
lactic  or  butyric  acids,  and  it  is  interesting  in  this  connection  to 
notice  that  according  to  Lindberger,^  the  former  of  these  two 
acids  exerts  a  distinctly  favouring  influence  on  tryptic  digestion, 
especially  in  presence  of  bile  and  sodium  chloride.  Thus  in 
presence  of  -02  p.  c.  lactic  acid  and  1  —  2  p.  c.  bile  and  sodium 
chloride  fibrin  may  be  digested  more  rapidly  than  in  a  neutral 
solution  and  fully  as  quickly  as  in  a  solution  of  moderate  alka- 
linity. But  the  presence  of  '05  p.  c.  of  lactic  acid  stops  the 
digestion. 

Traces  of  trypsin  have  been  stated  to  be  found  •  in  urine ;  this 
is  somewhat  doubtful.^ 

Trypsinogen. 

The  zymogen  of  trypsin.  Heidenhain  first  showed  that  the 
pancreas  contains,  in  its  absolutely  fresh  and  normal  condition, 
no  ready-made  enzyme,  but  an  antecedent  of  the  same.^  This 
body  is  readily  converted  into  the  active  enzyme  by  the  action  of 
dilute  acids  (1  c.c.  of  1  p.c.  acetic  acid  to  each  1  grm.  of  gland- 
substance)  and  a  conversion  also  takes  place  if  the  gland  is  kept 
for  some  time,  especially  in  the  warm,  this  resulting  most  prob- 
ably from  the  spontaneous  acidification  which  it  thus  undergoes. 
The  zymogen  is  soluble  in  strong  glycerine  without  conversion  into 
the  enzyme ;  it  is  also  soluble  in  water,  in  which  it  is  gradually 
changed  into  the  enzyme,  most  rapidly  when  warmed,  probably 
under  the  influence  of  the  acid  reaction  which  the  solution 
acquires.* 


Pialyn, 


In  addition  to  the  two  pancreatic  enzymes  which  have  already 
been  described,  both  the  secretion  and  the  gland-substance  contain 
a  third  substance  which  has  not  as  yet  been  isolated,  of  which, 
therefore,  but  little  is  known  from  a  chemical  point  of  view,  but 
which  must  be  regarded  as  an  enzyme  in  virtue  of  the  typical 
conditions  under  which  it  is  able  to  effect  a  hydrolytic  decompo- 

^  loc.  cit.  ref.  1,  on  p.  63. 
■^  For  litt.  see  ref.  1,  sub  Pepsin,  on  p.  61. 

3  Heidenhain,  Pfluger's  Arch.  Bd.  x.  (1875),  S.  581.     See  also  Podolinski,  Ibid. 
Bd.  xni.  (1876),  S.  422.     AVeiss,  Virchow's  Arch.  Bd.  lxviii.  (1876),  S.  413. 
*  Kuhne,  Lehrb.  d.  phi/siol.  Chem.  1868,  S.  120. 
^  From  TT7ap  =  fat,  and  \vfiu  =  to  split  up  or  decompose. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.         65 

sition  of  neutral  fats  into  glycerine  and  free  fatty  acid.  Bernard 
first  drew  attention  to  the  existence  of  this  enzyme.^  It  is  most 
actively  present  in  the  substance  of  the  fresh  gland  or  in  its  secre- 
tion, and  may  be  extracted  from  the  former  by  means  of  glycerine 
or  water.  In  every  case  it  is  essential  to  ensure  that  the  gland 
had  not  acquired  an  acid  reaction  before  extraction,  and  that  all 
acidification  in  the  extract  is  absent,  since  the  enzyme  is  pecu- 
liarly sensitive  to  acids  other  than  fatty,  and  is  readily  destroyed 
by  them. 2  Hence  a  dilute  alkaline  solution  should  be  employed, 
and  according  to  Paschutin  sodium  bicarbonate  mixed  with  the 
normal  carbonate  is  the  most  efficient  solvent.^ 

The  presence  of  the  enzyme  is  tested  for  by  adding  the  extract  to 
an  emulsion  of  oil  of  bitter  almonds,  or  other  neutral  oil  or  fat,  with 
gum  arabic;  the  mixture  is  then  most  carefullj^  neutralised  and  di- 
gested at  40°,  together  with  a  minimal  amount  of  neutral  sensitive 
litmus  solution.  In  presence  of  the  enzyme  the  mass  turns  more  or 
less  rapidly  red,  owing  to  the  liberation  of  the  free  fatty  acid. 

The  enzymic  nature  of  the  active  agent  is  shown  by  the  fact 
that  its  activity  is  greatest  at  about  40°,  is  destroyed  by  boiling, 
and  is  dependent  upon  the  reaction  of  the  digestive  mixture,  being 
greatest  in  presence  of  a  dilute  alkali,  although  it  will  show  itself 
in  a  neutral  solution.  It  will  also  be  observed  that  the  decom- 
position which  pialyn  effects  is  typically  hydrolytic. 

Rennin. 

Extracts  of  the  mucous  membrane  of  the  stomach  of  young 
animals,  and  more  especially  of  the  calf,  have  been  known 
from  time  immemorial  to  possess  a  most  remarkable  power  of 
causing  milk  to  clot,  and  rennet  was  commonly  employed  by  the 
Eomans  for  the  manufacture  of  cheese.  The  active  agent  in  pro- 
ducing the  clot  was  in  more  recent  times  supposed  to  be  either  the 
acidity  of  the  extract  itself  or  the  production  of  lactic  acid  from 
milk-sugar  (lactose)  by  means  of  some  active  principle  in  the 
extract.  Heintz  and  Hammarsten,  however,  showed  that  this 
view  is  untenable ;  and  we  now  know  that  the  substance  to 
which  the  clotting  is  due  is  an  enzyme  to  which  the  name  of 
'reniiin  may  be  conveniently  given.^  The  enzymic  nature  of  the 
active  agent  in  rennet  is  clearly  shown  by  the  typical  relationship 
which  it  exhibits  in  its  activity  to  the  reaction  of  the  solution  in 
which  it  is  present,^  to  the  temperature  at  which  its  activity  is 

1  Compt.  Rend.  T.  xxviii.  (1849),  p.  249.  See  also  his  Legons  de  physiol.  exper. 
T.  II.  (1856),  p.  253. 

2  Grutzner,  Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  302. 

3  Arch.  f.  Anat.  u.  Physiol.  Jahrg.  1873,  S.  386. 

*  This  name  seems  more  convenient  than  the  more  commonly  used  expressions 
'  the  rennet  ferment '  or  '  the  milk-curdling  ferment.' 

5  Hammarsten  (Swedish).  See  Abst.  in  Maly's  Bericht.  Bd.  ii.  (1872),  S.  121. 
Heintz,  Jn.f.  prakt.   Chem.  (N.F.)  Bd.  vi.  (1872),  S.  374.     See  also  Al.  Schmidt. 

5 


66  ENZYMES   OR   SOLUBLE  FERMENTS. 

greatest,  to  the  fact  that  the  briefest  exposure  to  100°  or  the  more 
prolonged  exposure  to  lower  temperatures  (70°  or  above)  ^  suffices 
to  destroy  its  active  properties,  and  to  the  fact  that  a  minute 
trace  suffices  to  clot  a  relatively  enormous  amount  of  casein.^ 

Nothing  is  known  as  to  the  chemical  nature  of  rennin.  Extracts  of 
the  gastric  mucous  membrane  contain  both  rennin  and  pepsin.  Ham- 
marsten  ^  has  obtained  it  free  from  the  latter  enzyme  by  fractional  pre- 
cipitation with  either  magnesium  carbonate  or  normal  lead  acetate,  by 
which  pepsin  is  more  readily  precipitated  than  is  rennin.  He  further 
endeavoured  to  separate  out  the  enzyme,  after  freeing  it  from  pepsin, 
by  precipitation  with  the  acetates  of  lead  in  presence  of  a  trace  of 
ammonia;  this  precipitate  was  then  carefully  decomposed  with  very 
dilute  sulphuric  acid,  and  the  enzyme  finally  separated  by  means  of 
cholesterin.  (Vide  preparation  of  pepsin,  p.  59.)  The  reactions  of 
the  purified  enzyme  described  by  Heiclenhain  seem  to  indicate  that  it 
is  not  a  proteid. 

Aqueous  and  glycerin  extracts  of  the  gastric  mucous  membrane 
are  usually  found  to  be  active  in  clotting  milk,*  but  the  activity 
of  a  faintly  acid  extract  is  in  all  cases  greater.  This  is  due  to  the 
existence  of  a  rennin  zymogen  (renninogen)  which  is  readily  con- 
verted into  tlie  enzyme  by  the  action  of  acids. ^  The  preparation 
of  highly  active  and  permanent  solutions  of  rennin  is  of  consider- 
able commercial  importance  in  connection  with  the  cheese-making 
industry.  .The  most  efficient  extractive  is  sodium  chloride,  5 — 15 
p.c. ;  and  permanency  is  attained  by  the  addition  of  alcohol,  or  in 
some  cases  thymol.^ 

Although  rennin  is  most  copiously  present  in  the  gastric  mucous 
membrane  of  the  calf,  it  may  be  obtained  from  the  tissue  of  almost 
any  stomach,  if  not  as  ready-made  enzyme,  at  least  in  the  form  of 
a  zymogen  (Hammarsten).  It  occurs  also  in  the  stomach  of  chil- 
dren ''  and  of  man  ;  ^  and  Roberts  has  described  a  similar  enzyme 
in  the  pancreas  of  the  pig,  ,ox,  and  sheep.^  Rennin  is  stated  to 
occur  in  traces  in  urine.  ^^ 

Maly's  Bericht.  Bd.  iv.  (1874),  S.  159.  Langley,  Jl.  of  Physiol.  Vol.  in.  (1881), 
p.  259. 

1  Mayer,  Die  Lehre  von  dem  chem.  Fermenten,  1882.  See  also  Maly's  Ber.  Bd.  x. 
(1880),  S.  208. 

^  400,000 — 800,000  times  its  own  weight.  Hammarsten.  See  Maly's  Bericht.  Bd. 
VII.  (1877),  S.  166. 

3  Maly's  Bericht.  Bd.  ii.  S.  121.  See  also  Friedberg,  Jl.  Amer.  Ch.  Soc.  May, 
1888,  p.  15. 

*  Hammarsten,  loc.  cit.  See  also  Erlenmeyer,  Sitzh.  d.  h.  b.  Akad.  d.  Wiss. 
Munchen,  1875,  Hft.  1. 

^  Hammarsten,  loc.  cit.     Langley,  Jl.  ofPhi/siol.  Vol.  in.  (1881),  p.  287. 

6  Soxhlet,  Milchzeitung,  1877,  Nos.  37,  38.  'Vhem.  Centralb.  1877,  S.  745.  Nessler, 
Landwirth.  Wochenblatt.  f.  Baden,  1882,  S.  57.  Friedberg,  Jl.  Amer.  Ch.  Soc.  May, 
1888,  p.  15.     Ringer,  Jl.  of  Physiol.  Vol.  xii.  (1891).     Note  2,  p.  164. 

■^  Zweifel,  Centralb.  f.  d.  med.  Wiss.  1874,  No.  59.  Hammarsten,  Lud wig's 
Festgabe,  Leipzig,  1875. 

8  Schumberg,  Virchow's  Arch.  Bd.  xcvii.  (1884),  S.  260.  Boas,  Centralb./.  d 
med.  Wiss.  1887,  No.  23. 

9  Proc.  Roy.  Soc.  No.  29,  1879,  p.  157. 

10  See  Helwes,  Pfliiger's  Arch.  Bd.  xliii.  (1888),  S.  384.  ' 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  67 

Fibrin-ferment. 

Buchanan's  work  (1831)  on  the  clotting  of  blood,  more  par- 
ticularly his  experiments  with  '  washed  clot,'  when  examined  in 
the  light  of  our  present  knowledge,  shows  clearly  that  he  was  in 
reality  dealing  with  that  factor  in  the  whole  process  which  was 
independently  discovered  by  Alexander  Schmidt  and  more  spe- 
cifically described  by  him  in  1872  under  the  name  of  '  fibrin-fer- 
ment.' ^  Its  existence  had  been  foreshadowed  in  some  experiments 
made  by  Brticke,  in  which  he  showed  that  the  fibrin oplastic  action 
of  precipitated  paraglobulin  was  partly,  at  least,  dependent  upon 
the  admixture  of  some  other  substance,  which  he  regarded  as  the 
truly  fibrinoplastic  factor.  Thus,  he  showed  among  other  things 
that  the  more  a  serum  is  diluted  before  the  paraglobulin  is  precipi- 
tated from  it  by  means  of  COg,  the  less  marked  are  its  fibrino- 
plastic powers. 2  Further,  Mantegazza  had  in  1871  put  forward 
the  view,  also  held  by  Buchanan,  that  the  white  corpuscles  play 
some  important  part  in  the  formation  of  fibrin,  without  in  any 
way  characterising  the  substance  which  he  suggested  was  prob- 
ably discharged  from  them  as  the  determinant  of  the  whole 
process.^  The  time  was  thus  ripe  for  Schmidt's  discovery.*  He 
prepared  the  ferment  by  precipitating  serum  with  15 — 20  vol- 
umes of  strong  alcohol ;  the  precipitate  was  treated  for  at  least 
14  days  with  the  alcohol  to  insure  complete  (?)  coagulation  and 
insolubility  of  the  proteids ;  after  which  time  it  was  removed  by 
filtration,  dried  in  vacuo  over  sulphuric  acid,  pulverised,  and 
extracted  with  distilled  water  in  volume  equal  to  twice  that  of 
the  serum  originally  employed.  The  ferment  solution  thus  ob- 
tained is  by  no  means  pure,  and  not  very  active.  More  recently 
Hammarsten  has  obtained  the  ferment  in  solution  free  from  para- 
globulin.^ He  saturates  serum  with  magnesium  sulphate  at  30°, 
and  filters  off  the  precipitated  paraglobulin  at  the  same  tempera- 
ture. The  filtrate  he  dilutes  with  nine  volumes  of  water,  and  to 
this  adds  gradually,  and  with  continuous  stirring,  dilute  caustic 
soda  until  a  permanent,  flocculent,  and  fairly  copious  precipitate 
is  formed.  This  precipitate  carries  the  ferment  down  mechani- 
cally, and  is  finally  washed,  pressed,  suspended  in  water,  dissolved 
by  acetic  acid  to  a  neutral  solution,  and  dialysed  till  free  from 
salt. 

For  ordinary  purposes  an  extremely  active  ferment  solution 
may  be  most  readily  obtained  by  Gamgee's  method  of  extracting 
the  so-called  '  washed  blood  clot  '  with  8  p.c.  solution  of  sodium 
chloride.^     The  solution  in  this  case  contains  a  large  amount  of 

'  An  account  of  Buchanan's  experiments  has  heen  given  by  Gamgee.  Physiol. 
Chemistry,  Vol.  i.  1880,  p.  43.     See  also  .//.  of  Physiol.  Vol.  ii.  (1879),  p.  145. 

2  Sitzb.  d.  Wien.  Akad.  Bd.  lv.  (2  Abth.)',  1867,  S.  891. 

3  See  Ahst.  in  Maly's  Bericht.  Bd.  i.  (1871),  S.  110. 
*  Pfluger's  Arch.  Bd.  vi.  (1872),  S.  457. 

5  Ibid.  Bd.  XVIII.  (1878),  S.  89;  xxx.  (1883),  S.  457. 

6  Gamgee,  Jl.  of  Physiol.  Vol.  ii.  (187,9),  p.  150. 


68  ENZYMES   OR   SOLUBLE  FERMENTS. 

globulins  in  solution,  as  also  does  the  similar  extract  which  may 
be  equally  efficiently  prepared  from  ordinary  washed  fibrin. ^ 

In  no  case  as  yet  has  the  fibrin-ferment  been  obtained  in  a  con- 
dition of  such  purity  as  to  justify  any  dogmatic  statement  as  to  its 
chemical  composition.  All  the  solutions  whose  preparation  has  been 
described  above  yield  strong  proteid  reactions,  and  Halliburton  ^ 
has  argued  from  his  own  experiments  and  a  criticism  of  preceding 
work  that  the  ferment  is  really  a  proteid  identical  (?)  with  what 
he  had  previously  called  '  cell-globulin '  (antea,  p.  28).  On  the 
other  hand  it  is  possible  by  appropriate  methods  to  free  the  salt- 
extracts  of  fibrin  very  completely  from  proteids  without  any  great 
loss  of  ferment  activity,  certainly  without  any  such  loss  as  would 
necessarily  be  the  case  if  the  active  substance  were  a  globulin.^ 
It  may  be  said  that  the  apparent  ferment-powers  in  such  cases 
are  in  reality  due  to  the  presence  of  calcium  sulphate,  which  is 
now  known  to  promote  the  clotting  of  a  dilute  salt-plasma  to  an 
extraordinary  degree ;  ^  but  as  against  this  the  fact  may  be  quoted 
that  solutions  free  from  proteid  reaction,  and  which  had  been 
freed  from  salts  by  careful  dialysis,  lost  their  activity  on  heating 
to  60 — 70°,  which  they  would  not  have  done  had  the  activity 
been  due  merely  to  calcium  sulphate. 

When  Schmidt's  method  is  applied  to  blood  received  directly 
from  an  artery  into  an  excess  of  alcohol  no  ferment  can  be  ob- 
tained from  the  precipitate  thus  obtained.  It  is  hence  evident 
that  the  living,  circulating  blood  contains  no  preformed  ferment, 
and  the  question  thus  arises  from  what  does  it  take  its  origin 
during  the  clotting  of  blood  and  ]3i"esumably  as  an  immediate 
antecedent  to  that  clotting  ?  Buchanan  held  distinctly  the  view 
that  the  active  agent  in  the  whole  process  was  in  some  way  con- 
nected with,  if  not  derived  from,  the  white  corpuscles,  a  view  also 
held  later  on  by  Mantegazza.  Schmidt  also  took  this  view,  bas- 
ing it  on  an  elaborate  series  of  investigations  for  which  his  orig- 
inal works  must  be  consulted.^  Lowit,  experimenting  with 
lymph  as  well  as  blood,  while  denying  that  the  white  corpuscles 
break  down  at  clotting  in  the  way  Schmidt  described,  still  connects 
them  with  the  production  of  the  initiative  factor  in  the  whole 
process.^  Still  further  evidence  in  the  same  direction  may  be 
derived  from  the  experiments  of  Eauschenbach  "  and  Halliburton,^ 
and  of  Fano,  who  observed  that  when  peptone-plasma  is  freed  as 

1  Lea  aud  Green,  Jl.  of  Phi/siol.  Vol.  iv.  (1883),  p.  386, 

2  Jl.  ofPhijsiol.  Vol.  ix.  (1888),  p.  265. 

3  Lea  and  Green,  loc.  cit. 

*  Green,  Ibid.  Vol.  vm.  (1887),  p.  354. 

5  Pfliiger's  Arch.  Bd.  ix.  (1874),  S.  353;  xi.  (1875),  Sn.  291,  515. 

6  Sitzb.  d.   Wien.  Akad.  (2  Abth.),  Bd.  lxxxix.  (1884),  S.  270;  xc.  S.  80. 

■^  Inaug.-Diss.  Dorpat,  1883.  See  also  the  Dissertations  (Dorpat)  of  F.  Hoffmann, 
1881.     Samson-Himmelstjerna,  1882;  Heyl,  1882. 

8  loc.  cit.     See  also  Kriiger,  Zt.f.  Biol.  xxiv.  (1888),  S.  189. 


CHEMICAL   BASIS   OF  THE  ANIMAL   BODY.  69 

completely  as  possible  from  white  corpuscles  it  cannot  be  made  to 
clot  in  the  usual  way  by  the  addition  of  water.  ^ 

In  addition  to  the  red  and  white  corpuscles  blood  also  contains,  as 
already  described  (§  33),  a  third  structural  element,  the  'platelets,' 
and  several  observers  have  endeavoured  to  connect  the  first  cause  of 
the  clotting  of  blood  with  some  breaking  down  and  disappearance  of 
these  structures.^  This  view  is  as  yet  insufficiently  supported,  and  is 
combated  by  several  observers ;  3  bearing  in  mind  however  how  little 
is  known  about  the  origin  and  nature  of  these  platelets  the  question 
of  their  relationship  to  blood-clotting  must  still  be  regarded  as  await- 
ing a  decisive  answer. 

In  addition  to  the  undoubted  relationship  of  leucocytes  to 
fibrin-formation  it  appears  that  the  protoplasm  of  many  other 
cells,  both  animal  and  vegetable,  may  exert  an  influence  similar 
to  that  of  the  white  corpuscles  of  blood.* 

Wooldridge  regarded  the  leucocytes  as  entirely  secondary  and  very 
subordinate  factors  in  the  process  of  clotting,  as  also  the  fibrin-fer- 
ment. According  to  his  view  blood-plasma  contains  in  itself  all  the 
elements  requisite  for  the  formation  of  fibrin,  which  he  considers  to 
be  in  no  sense  the  outcome  of  any  fermentative  process.  He  described 
three  coagulable  proteids  A-  S-  and  C-fibrinogen.  The  last  of  these 
occurs  in  minimal  quantities  in  plasma,  is  identical  with  the  sub- 
stance ordinarily  known  as  fibrinogen,  and  clots  on  the  addition  of 
fibrin-ferment.  According  to  his  view  clotting  is  due  to  a  transfer- 
ence of  lecithin  from  its  combination  Avith  ^-fibrinogen  to  ^-fibrino- 
gen, by  which  means  both  the  fibrinogens  disappear  and  fibrin  takes 
their  place.  ^ 

The  information  which  we  possess  as  to  the  nature  of  the 
fibrin-ferment  is  much  less  complete  and  satisfactory  than  in  the 
case  of  other  enzymes.  But  that  it  is  properly  placed  in  the  class 
of  these  substances  is  shown  by  the  typical  facts  that  its  activity 
is  closely  dependent  upon  temperature,  being  destroyed  by  heat- 
ing to  70°  ;  that  it  does  not  affect  the  amount  but  only  the  rate 
of  change  of  fibrinogen  into  fibrin  ;  that  it  is  carried  down  by 
gelatinous  precipitates  formed  in  its  solutions  (Hammersten),  pro- 
duces a  change  which  is  out  of  all  proportion  to  the  mass  of 

1  Arch.  f.  PhijsioL  Jahrg.  1881,  S.  288.     Centralb.  f.  d.  med.  Wiss.  1882,  S.  210. 

2  Hayein,  Gaz.  med.  de  Paris,  1878,  p.  107.  Compt.  Rend.  T.  lxxxvi.  (1878), p. 
.58.  Arch,  de  Physiol.  1878,  p.  692.  Bizzozero,  Virchow's  Arch.  Bd.  xc.  (1882),  S. 
261.  Laker,  Sitzb.  d.  Wien.  Akad.  (2  Abth.),  Bd.  lxxxvi.  (1883),  S.  173.  Hayem's 
colourless  ' hasmatoblasts '  are  identical  with  Bizzozero's  'platelets.'  The  true 
hseniatohlasts  are  the  cells  described  by  Neumanu,  Rindfieisch,  aud  others  as  occur- 
ring in  the  red  marrow  of  bones. 

^  Fano,  Centralb.  f.  d.  med.  Wiss.  1882,  S.  210.  Lei  wit,  loc.  cit.  Schimmelbusch, 
Virchow's  Arch.  Bd.  ci.  (188.5),  S.  201. 

*  Rauschenbach,  loc.  cit.     Grohniann,  Inaug.-Diss.  Dorpat,  1884. 

5  Croonian  Lecture,  Roy.  Soc.  Lond.  1886.  Ludwig's  Festschrift,  1887.  See  also 
Halliburton,  loc.  cit.  antea. 


70  ENZYMES   OR   SOLUBLE   FERMENTS. 

enzyme  employed,  and  is  not,  so  far  as  we  know,  used  up  in  the 
change  which  it  induces,  since  it  is  present  in  serum. 

Muscle  enzyme. 

The  phenomena  of  the  clotting  of  muscle-plasma  compared  with 
those  of  blood-plasma  and  the  relationship  of  the  process  to  the 
presence  of  neutral  salts  and  to  temperature  suggest  at  once  that 
the  change  is  probably  one  in  which  some  enzyme  plays  a  part. 
Immediately  after  Schmidt's  discovery  of  the  fibrin-ferment  the 
question  of  the  existence  of  a  myosin-ferment  was  investigated 
under  his  guidance,^  and  resulted  in  the  discovery  of  the  exist- 
ence in  muscles  of  an  enzyme  which  appeared  to  be  identical 
with  fibrin-ferment  rather  than  specifically  myosinic.  The  later 
work  of  the  Dorpat  School  further  confirmed  the  above,  but  failed 
to  establish  the  existence  of  an  enzyme,  differing  from  fibrin-fer- 
ment and  specifically  active  in  promoting  the  clotting  of  muscle- 
plasma.  ^  More  recently  it  has  been  shown  that  by  applying 
Schmidt's  method  to  muscles  which  have  been  treated  for  some 
time  with  alcohol,  a  solution  may  be  obtained  which  hastens  the 
clotting  of  diluted  muscle-plasma,  does  not  facilitate  the  forma- 
tion of  fibrin  in  blood-plasma,  and,  unlike  fibrin-ferment,  requires 
to  be  heated  to  100°  before  it  loses  its  activity .^  The  active 
asent  in  the  solution  is  therefore  not  identical  with  fibrin-ferment 
and  may  be  spoken  of  as  a  myosin-ferment. 

Urea-ferment. 

When  urine  is  exposed  to  the  air  its  acidity  at  first  increases, 
but  in  most  cases  this  speedily  gives  way  to  a  marked  alkalinity, 
which  is  accompanied  by  the  evolution  of  ammonia.  This  is  due 
to  a  hydrolytic  fermentative  change  resulting  from  the  appear- 
ance and  development  in  the  urine  of  certain  micro-organisms  of 
which  the  best  known  is  the  Torula  ureae.*  Normally  urine  is 
free  from  these  organisms  and  may  be  kept  in  the  excised  blad- 
der for  an  indefinite  period  without  exhibiting  any  tendency  to 
become  alkaline  ;  ^  in  certain  abnormal  conditions  it  may  undergo 
an  active  alkaline  fermentation  while  still  in  the  bladder.  The 
part  played  by  the  organisms  was  for  a  long  time  regarded  as 
similar  to  that  of  yeast-cells  in  promoting  alcoholic  fermentation. 
Soon  however  evidence  was   adduced   which   showed   that   the 

1  Michelson,  Diss.  Dorpat,  1872. 

2  Grubert,  Diss.  Dorpat,  1883.     Klemptner,  Ihid.     Kugler,  Ibid. 

3  Halliburton,  Jl.  of  Physiol.  Vol.  vm.  (1887),  p.  159. 

*  Miiller,  Jn.  f.  prakt.  Chem.  Bd.  lxxxi.  (1860),  S.  467.  Pasteur,  Compt.  Rend. 
T.  L.  1860,  p.  869.  van  Tieghem,  Ihid.  T.  lviii.  1864,  p.  210.  But  see  also  Jaksch, 
Zt.  f.physiol.  Chem.  Bd.  v.  (1881),  S.  395.  Leube,  Yirchow's  Arch.  Bd.  c.  (1885), 
S.  540.  'Miquel,  Bidl.  de  la  Soc.  Chim.  T.  xxix.  (1878),  p.  387;  xxxi.  p.  391; 
XXXII.  (1879),  p.  126. 

5  Cazeneuve  et  Livon,  Compt.  Rend.  T.  lxxxv.  (1877),  p.  571.  Bull,  de  la  Soc. 
Chim.  T.  xxviii.  (1877),  p.  484. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.         71 

change  was  not  necessarily  due  solely  to  the  life  and  growth  of 
the  organisms  in  the  solution,  for  it  was  found  that  the  fermenta- 
tion might  be  very  complete  in  presence  of  an  amount  of  carbolic 
acid  which  is  fatal  to  the  development  of  micro-organisms.^  The 
probable  existence  of  an  enzyme  as  a  possible  factor  in  the  whole 
process  which  was  thus  demonstrated  was  reduced  to  a  certainty 
by  the  experiments  of  Musculus.^  Employing  the  thick  mucous 
excretion  of  urinary  catarrh  he  precipitated  the  mucin  with  al- 
cohol, dried  the  precipitate  at  a  low  temperature,  extracted  it 
with  water  and  found  the  extract  to  possess  active  hydrolytic 
powers  in  a  solution  of  urea.  The  proof  of  the  existence  of  the 
enzyme  in  a  pathological  mucous  urine  in  which  there  is  fre- 
quently no  reason  to  suspect  the  existence  of  any  micro-organisms 
still  left  open  the  question  of  the  isolation  of  the  enzyme  from 
the  micro-organism  itself.  When  urine  which  by  exposure  to 
the  air  has  entered  into  active  alkaline  fermentation  and,  as 
shown  by  microscopic  examination,  is  full  of  Torulae,  is  efficiently 
filtered  no  enzyme  capable  of  hydrolising  urea  can  be  precipitated 
by  alcohol  from  the  clear  filtrate.  If  on  the  other  hand  the  unfil- 
tered  urine  be  precipitated  with  an  excess  of  alcohol  and  the  pre- 
cipitate washed  with  alcohol  and  dried  in  the  air,  a  powder  is 
obtained  which  is  itself  extraordinarily  active,  and  yields  to  an 
aqueous  extract  a  soluble  enzyme  which  rapidly  converts  urea 
into  ammonia  and  carbonic  acid.  The  rapidity  of  the  conversion 
precludes  the  intervention  of  any  developing  organism,  and  that 
the  change  is  truly  due  to  an  enzyme  is  shown  by  the  fact  that  it 
takes  place  with  equal  readiness  in  presence  of  chloroform.^ 

It  is  of  some  interest  to  notice  here  that  from  what  has  been  said 
above  the  organisms  to  Avhose  activity  the  fermentation  is  due  do  not 
discharge  their  enzyme  into  the  surrounding  medium;  when  killed 
however,  as  by  means  of  alcohol,  they  yield  it  readily  to  a  suitable 
extractive.  This  holds  good  also  in  the  case  of  invertin,  which  is  not 
found  in  the  filtrate  from  yeast,  while  it  may  readily  be  extracted  from 
the  cells  when  killed  by  ether  or  alcohol.*  Similarly  it  appears  that 
putrefactive  bacteria  may  excrete  or  yield  an  enzyme  whose  action  is 
closely  analogous  to  that  of  trypsin.^ 

The  most  prolific  source  of  the  urea  enzyme  is  in  all  cases  the 
mucous  urine  passed  in  inflammatory  conditions  of  the  bladder. 

1  Hoppe-Seyler,  Med.-chem.  Untersnch.  Hit.  4,  1871,  S.  .570. 

2  Compt.  Rend.  T.  lxxviii.  (1874),  p.  132;  lxxxii.  (1876),  p.  334.  Pfliiger's 
Arch.  Bd.  xii.  (1876),  S.  214.     See  also  Lailler,  Compt.  Rend.  T.  lxxviii.  p.  361. 

a  Lea,  JL  of  Physiol.  Vol.  vi.  (1885),  p.  136. 

*  Hoppe-Seyler,  Ber.  d.  dentsch.  cliem.  Gesell.  1871,  S.  810.  Confirmed  by  Lea 
For  chemistry  of  invertin  see  Dona.th,  Ber.  d.  deutsck.  chem.  Gesell.  1875,  S.  795; 
1878,  S.  1089.  Earth,  Ibid.  1878,  S.  474.  Kjeldahl  (Danish).  See  Abst.  in  Maly's 
Bericht.  1881,  S.  448.  Mayer,  Zt.  f.  Spirit-lndust.  1881,  Nos.  16,22.  Low,  Tfluger's 
Arch.  Bd.  XXVII.  (1882),  S.  203. 

5  Hiifner,  Jn.f.prakt.  C/zem.  (N.F.)  Bd.  v.  (1872),  S.  372.  Herrmann,  Zt.f. 
physiol.  Chem.  Bd.  xi.  (1887),  S.  523.  E.  Salkowski,  Zt.  f.  Biol.  Bd.  xxv.  (1889), 
S.  92. 


72  ENZYMES   OR  SOLUBLE  FERMENTST^ 

In  this  case  the  enzyme  appears  to  be  closely  associated  with  the 
mucin  and  is  presumably  a  secretory  product  of  the  mucous  mem- 
brane, for  it  is  frequently  obtained  when  there  has  been  no  opera- 
tive use  of  surgical  instruments  which  could  account  for  the  intro- 
duction of  micro-organisms  from  the  exterior. 


In  concluding  this  account  of  the  more  important  enzymes  of 
the  animal  body  it  may  not  be  out  of  place  to  say  a  few  words  on 
the  probable  mode  of  action  of  the  ferments  and  enzymes. 

The  term  fermentation  was  applied  originally  to  the  changes, 
accompanied  by  characteristic  frothing,  foaming,  and  evolution  of 
gases,  which  saccharine  solutions  such  as  the  expressed  juice  of 
fruits  or  infusions  of  grain  undergo  on  exposure  to  the  air.  The 
chemical  changes  and  products  of  the  fermentation  were  studied 
from  the  earliest  times,  and  in  1680  Leuwenhcek  described,  with 
the  aid  of  the  newly-invented  microscope,  the  small,  spherical  par- 
ticles which  are  now  known  as  yeast-cells,  to  be  the  exciting  cause 
of  the  whole  process.  He  did  not  however  ascribe  any  organisa- 
tion to  these  particles,  and  it  was  not  until  1835  and  1837  that 
Cagniard  de  Latour  and  Schwann  respectively  but  independently 
took  up  the  investigation  where  Leuwenhcek  had  left  it,  and  estab- 
lished firmly  and  finally  the  organised  and  plant-like  nature  of  the 
yeast-cell  and  the  absolute  dependence  of  fermentation  upon  its 
presence  in  the  fermenting  fluid. ^  The  yeast-cell  having  thus  been 
definitely  recognized  as  the  cause  of  the  fermentation,  the  interest- 
ing question  at  once  arose  as  to  how  the  known  cause  produces 
the  observed  effect,  and  to  this  question  many  answers  have  been 
given,  of  which  the  following  are  the  more  important. 

Liebig  regarded  the  ferments  as  substances  in  a  state  of  pro- 
gressing decomposition  during  which  the  equilibrium  of  their 
constituents  is  upset  and  a  rapid  motion  of  their  minuter  parts 
established.  When  brought  into  contact  with  other  decomposable 
substances  the  motion  of  the  ferment's  particles  is  communicated 
to  the  former,  whereupon  it  also  undergoes  a  decomposition  result- 
ing in  the  formation  of  the  simpler  products  which  make  their 
appearance  and  are  characteristic  of  the  fermentation.  According 
to  this  view  the  organised  nature  of  the  yeast-cells  is  left  out  of 
account  and  the  phenomena  attributed  entirely  to  the  purely 
chemical  decomposition  of  their  constituent  substance,  set  going 
at  the  outset  by  oxygen.^     Pasteur  regarded  alcoholic  fermenta- 

1  Erxleben  in  1818  had  described  and  spoken  of  yeast  as  a  vegetative  organism, 
as  also  in  1825  had  Desmazieres,  who  ascribed  to  it  an  animal  rather  than  vegetable 
nature. 

2  Ann.  d.  Chem.  u.  Pharm.  Bd.  xxx.  (1839),  Sn.  250,  363.  Stahl  in  1734  had  ex- 
pressed practically  identical  views. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  73 

tion  as  indissolubly  connected  with  the  vegetative  growth,  multi- 
plication, and  metabolism  of  the  yeast-cell.  According  to  this  view 
sugar  is  the  food-stuff  out  of  which  the  organism  obtains  the  ma- 
terial  requisite  for  its  metabolism  and  growth,  the  products  of  the 
fermentation  being  thus,  as  it  were,  the  excretionary  residues  of 
the  metabolised  food.^  A  third  view  attributes  the  fermentative 
decomposition  to  the  production  by  the  organised  ferments  of  solu- 
ble unorganised  enzymes  to  whose  activity  the  decomposition  is 
due.  This  view  received  its  chief  support  from  the  discovery  that 
a  part  at  least  of  the  change  which  sugar  undergoes  in  presence  of 
yeast  may  be  obtained  by  means  of  the  soluble  enzyme  '  invertin ' 
which  can  readily  be  extracted  from  the  dead  cells.''^  But  as  yet 
all  efforts  to  obtain  an  enzyme  capable  of  carrying  the  decomposi- 
tion beyond  the  initial  stage  of  inversion  have  been  fruitless.  Ac- 
cording to  von  Nageli  the  living  substance  of  the  organised  cell  is 
to  be  regarded  as  being  in  continuous  and  rapid  molecular  vibra- 
tion, and  the  decomposition  of  the  fermentable  substance  as  the 
result  of  the  direct  transference  of  these  vibrations  to  this  sub- 
stance, by  means  of  which  its  equilibrium  is  upset  and  it  is  split 
up  into  simpler  and  therefore  more  stable  products.^  To  discuss 
the  merits  of  these  various  theories  and  the  experiments  upon 
which  they  are  based  is  quite  impossible  within  any  reasonable 
limits  of  brevity.  We  shall  perhaps  be  not  far  wrong  in  consider- 
ing that  as  regards  the  organised  ferments  the  changes  they  effect 
may  be,  in  their  earlier  stages,  partly  the  outcome  of  the  action  of 
some  soluble  enzyme,  and  partly  the  result  of  that  cycle  of  meta- 
bolic (chemical)  processes  which  occur  continuously  in  their  proto- 
plasm, in  virtue  of  which  they  are  spoken  of  as  '  living.'  Simi- 
larly in  the  higher  animals  we  find  a  large  number  of  simpler 
processes  carried  on  by  means  of  isolable  enzymes,  by  which  un- 
doubtedly the  labours  of  the  protoplasm  in  performing  its  own 
more  complicated  activities  are  materially  lightened.  But  we  are 
still  face  to  face  with  numberless  decompositions  which  cannot  as 
yet  be  reproduced  outside  the  limits  of  living  matter  and  which 
cannot  be  explained  with  reference  to  anything  other  than  the 
direct  activity  of  living  matter. 

The  general  conditions  and  factors  which  characterise  the  action 
of  the  soluble  ferments  or  enzymes  have  already  been  mentioned 
(p.  53),  but  without  making  any  suggestion  as  to  the  probable  way 
in  which  they  produce  and  carry  on  the  decompositions  to  which 
they  give  rise.  Liebig's  theory  of  the  mode  of  action  of  yeast, 
since  it  left  the   organisation  and  life  of  the  cell  entirely  out  of 

1  This  view  was  keenly  attacked  by  Liebig,  Ann.  d.  Chem.  u.  Pharm.  Bd.  CLiii. 
(1870),  Sn.  1,  137.  See  Pasteur  in  reply,  Ann.  Chim.  Phijs.  4  Se'r.  T.  xxv.  (1872), 
p.  145. 

2  The  inverting  power  of  yeast  was  first  stated  by  Dubrunfaut  in  1847.  Berthelot 
obtained  invertin  in  solution  in  1860,  and  Hoppe-Seyler  prepared  it  in  the  form  of  a 
soluble  powder  in  1871.     See  references  on  p.  71. 

^  Theorle  der  Gdhrung,  Miinchen,  1879. 


74  ENZYMES   OR   SOLUBLE   FERMENTS. 

account,  and  was  based  simply  upon  the  supposed  properties  of 
the  changing  cell-substance,  might  obviously  therefore  be  applied 
to  any  ordinary  soluble  enzyme.  There  is  however  no  evidence  to 
show  that  the  enzymes  are  in  the  state  of  change  or  decomposi- 
tion which  Liebig  supposed ;  on  the  contrary  they  are  observed  to 
be  on  the  whole  remarkably  stable  substances,  from  the  point  of 
view  that  a  minute  trace  can  produce  a  profound  decomposition  in 
a  relatively  enormous  mass  of  material,  during  an  almost  indefi- 
nitely long  time,  without  itself  undergoing  any  proportionate  altera- 
tion or  destruction.!  The  theory  of  v.  Nageli  previously  quoted 
-•■/as  applied  by  its  author  to  explain  the  fermentative  power  of 
the  living  cell,  and  is  thus  not  directly  applicable  to  the  non-living 
enzymes.  Mayer,  it  is  true,  has  put  forward  a  view  which  is  essen- 
tially a  development  of  v.  Nageli's  and  is  applicable  to  the  en- 
zymes. These  substances  are  in  all  cases  produced  solely  and 
entirely  by  the  activity  of  living  cells  or  organisms,  and  Mayer 
regards  them  as  retaining  in  themselves  a  portion  of  that  molecu- 
lar motion  which  is  supposedly  so  characteristic  of  the  living 
parent  cell  from  which  they  have  been  separated.^  It  cannot  how- 
ever be  said  that  these  theories  afford  any  real  insight  into  the 
probable  mode  of  action  of  an  enzyme,  and  we  must  look  for  it  in 
some  other  direction. 

Attention  has  been  already  drawn  (p.  53)  to  the  existence  of  a 
large  and  increasing  class  of  chemical  reactions  whose  occurrence 
is  determined  by  mere  traces  of  some  substance  which  does  not 
itself  at  the  same  time  undergo  any  change  during  the  decomposi- 
tions which  it  initiates,  and  the  enzymes  have  been  compared  to 
these  substances.  Now  in  the  case  of  the  reactions  of  which  we 
are  now  speaking  it  is  known  in  some  and  probable  in  all  that  the 
process  which  takes  place  is  in  general  terms  the  following.  The 
determinant  substance  interacts  with  one  of  the  reagents  to  form 
a  compound  which  can  now  enter  into  combination  with  the  other , 
the  result  is  the  formation  of  a  more  complex  compound  which  at 
once  decomposes,  giving  rise  to  products  of  which  one  is  the  origi- 
nal determinant  substance  in  an  unaltered  form,  the  others  the 
product  characteristic  of  the  reaction. ^  This  suggests  at  once  that 
the  enzymes  may  play  their  part  in  a  manner  similar  to  that  of 
the  determinant  in  the  above  reactions,  a  view  which  has  been  put 
forward  but  scarcely  receives  the  attention  that  it  deserves.* 

1  Berzelius  explained  fermentation  as  the  outcome  of  a  mysterious  '  catalytic  ac- 
tion,' or  'action  by  presence '  or  '  contact.'  He  tiius  compared  ferments  to  platinum- 
black,  which  is  able,  in  minute  quantity,  to  cause  a  liberation  of  oxygen  from  peroxide 
of  hydrogen  without  itself  undergoing  any  recognisable  change.  Tliis  is  however  no 
explanation,  for  it  does  not  amount  to  more  than  saying  that  given  the  contact  of 
two  substances  capable  of  reacting  on  each  other,  a  certain  reaction  takes  place. 

'^  Die  Lehre  von  den  cliem.  Fermenten,  Heidelb.  1882. 

'^  Vide  the  reactions  in  the  contLnuous  etherification  process  and  the  manufacture 
of  sulphuric  acid.  See  also  Traube  (Ber.  d.  deutsch.  chem.  Gesell.  1885,  S.  1890),  on 
the  part  played  by  water  in  determining  the  explosion  of  O  and  CO. 

*  Kiihne,  Lehrb.  d.physwl.  Chem.  1868,  S.  39.     Hoppe-Seyler,  Med.-chem.  Unters. 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.  75 

In  most  cases  it  is  known,  and  it  is  probable  in  all,  that  the 
soluble  ferments  act  by  bringing  about  a  union  of  water  with  the 
substances  upon  which  they  act.  This  process  might  be  supposed 
to  take  place  in  the  following  way.  The  enzyme  uniting  with  the 
substance  to  be  decomposed,  the  compound  thus  formed  is  now 
able  to  unite  with  water,  and  this  final  more  complex  and  hence 
less  stable  compound  undergoes  a  decomposition  of  which  the 
original  enzyme  is  one  product,  the  others  being  the  hydrated  and 
hence  altered  substance  whose  formation  is  characteristic  of  the 
whole  process.  It  is  impossible  within  convenient  limits  to  bring 
forward  here  all  the  direct  evidence  in  favour  of  the  above  view 
as  to  the  mode  of  action  of  enzymes ;  it  must  suffice  to  say  that  as 
regards  pepsin  there  is  some  reason  for  thinking  that  it  can  enter 
into  combination  with  hydrochloric  acid.  Finally  it  may  be  stated 
that  the  characteristic  phenomena  of  zymolysis  in  connection  with 
the  influence  of  heat,  the  effect  of  various  salts  and  dilution,  the 
cessation  of  the  change  in  presence  of  an  excess  of  the  products  of 
that  change,  &c.,  are  such  as  careful  consideration  shows  might 
from  several  points  of  view  be  expected  on  the  supposition  that 
the  above  theory  of  enzyme  action  is  true. 

Chemical  action  is  in  all  cases  accompanied  bj^  an  evolution  or  ab- 
sorption of  heat,  and  it  will  add  to  the  completeness  of  this  account  of 
the  ferments  if  we  consider  bnefl}^  the  heat-phenomena  which  accompany 
the  chemical  action  due  to  the  enz3anes.  Liebig  regarded  the  fermenta- 
tive decomposition  of  sugar  as  necessitating  a  considerable  consumption 
of  energy^  which  he  supposed  to  be  derived  from  the  decomposing  albu- 
min of  the  ferment-substance.  Hoppe-Seyler  on  the  other  hand  put 
forward  the  general  view  that  heat  is  evolved  in  every  case  of  ferment 
action,  basing  it  upon  experiments  in  which  he  observed  a  distinct 
rise  of  temperature  during  the  action  of  pancreatic  extracts  upon 
starch,  but  more  particularly  upon  the  opinion  that  the  heat  of  com- 
bustion of  the  products  of  zymolysis  is  in  all  cases  less  than  that  of 
the  original  substance  from  which  the  products  have  been  formed.-^ 
And  this  is  undoubtedly  the  correct  view.  In  addition  to  Hoppe- 
Seyler  other  observers  have  observed  a  rise  of  temperature  during 
zjanolysis,  e.  g.  in  the  case  of  the  formation  of  fibrin,^  the  clotting 
of  miik,^  and  the  inversion  of  cane-sugar.'*  Mai}"-  on  the  other  hand 
observed  a  considerable  absorption  of  heat  during  the  action  of  pepsin 
on  proteids  and  ptyalin  on  starch.^  These  experiments  it  may  be  ob- 
served are  discordant,  and  in  reality  they  neither  speak  strongly  for 
nor  against  the  evolution  of  heat  during  the  action  of  the  enzymes ;  as 

Hft.  4,  1871,  S.  573.     v.  Wittich,  Pfliiger's  J.rcA.  Bd.  v.  (1872),  S.  435.  AVurtz,  Compt. 
Rend.  T.  xci.  (1880),  p.  787. 

1  Mecl.-Chem.  Unters.  Hft.  4,  1871,  S.  574. 

2  Le'pine,  Gaz.  Med.  Paris,  1876.  No.  12. 

3  Mayer,  Milchzeitunij,  1881,  No.  2,  3,  4,  6.     See  Abst.  in  Maly's  Bericht.  1880,  S. 
209.     But  see  also  Musso,  Ibid.  1879,  S.  16. 

*  Kunkel,  Pfliiger's  Arch.  Bd.  xx.  1879,  S.  509      But  see  Nageli,  Ibid.  Bd.  xxii. 
S.  310. 

5  Pfluger's  Arch.  Bd.  xxii.  (1880),  S.  111. 


76  MUCIN. 

a  matter  of  fact  they  could  scarcely  be  expected  to  do  so,  since  it  is  ex- 
tremely difficult  to  make  allowance  for  the  heat  which  may  he  simply 
absorbed  or  set  free  as  the  result  of  the  varying  solubilities  of  the  orig- 
inal substance  and  the  products  of  its  decomposition.  The  real  proof 
of  the  correctness  of  Hoppe-Seyler's  view  is  the  fact,  already  stated, 
that  the  heat  of  combustion  of  the  products  of  zymolysis  is  less  than 
that  of  the  substance  from  which  they  are  derived.^ 


NlTEOGENOUS   NoN-CRYSTALLINE    BODIES    ALLIED    TO    PEOTEIDS. 

These  resemble  the  proteids  in  many  general  points,  but  exhibit 
among  themselves  much  greater  differences  than  do  the  proteids. 
As  regards  their  molecular  structure  nothing  satisfactory  is  known. 
Their  percentage  composition  approaches  that  of  the  proteids,  and 
like  these  they  yield,  under  hydrolytic  treatment,  large  quantities 
of  leucin  and  in  some  cases  tyrosin.     They  are  all  amorphous. 

Mucin. 

This  is  the  substance  which  gives  to  many  animal  secretions, 
such  as  saliva,  bile,  synovial  fluid,  &c.,  their  characteristic  ropy 
consistency.  It  may  also  be  obtained  by  the  use  of  appropriate 
solvents  from  the  tissues  themselves,  such  as  submaxillary  gland, 
tendons,  and  umbilical  cord.  It  is  peculiarly  copious  in  the  secre- 
tion which  may  be  collected  on  stimulating  the  mantle  of  Helix 
pomatia,  or  in  an  extract  of  the  tissues  of  this  animal.  The  gen- 
eral phenomena  of  the  formation  of  mucin  by  mucous  cells,  and 
more  particularly  the  characteristic  behaviour  of  the  mucous 
granules  in  relation  to  the  secretory  activity  of  the  sub-maxillary 
gland,^  leave  but  little  doubt  that  mucin  is  to  be  regarded  as  de- 
rived from  the  true  proteids;  in  conformity  with  this  it  yields 
many  of  the  reactions  characteristic  of  the  proteids  (Millon's  and 
xanthoproteic),  and  by  the  action  with  mineral  acids  some  form 
of  acid-albumin  is  usually  obtained.  During  this  treatment  (or 
with  alkalis)  moreover  a  second  product  generally  makes  its  ap- 
pearance, which  belongs  to  the  group  of  carbohydrates  and  by 
heating  with  acids  may  be  made  to  yield  a  reducing  sugar.  Not- 
withstanding the  views  which  have  frequently  been  advanced 
that  mucin  is  in  reality  a  mixture  of  proteid  and  carbohydrate 
material,  it  is  now  known  with  considerable  certainty  that  it  is 
a  unitary  substance  which,  from  what  has  been  already  said, 
might  be  almost  regarded  as  an  animal   glucoside.      It  further 

1  For  heat  of  combustion  of  physiologically  important  substances  see  Eechenberg. 
[naug.  Diss.  Leipzig,  1880,  and  Jn.  f.  prakt.  Chem.  (N.  F.)  Bd.  xxii.  (1880),  Sn.  1, 
223.  See  also  Stohmann,  Ibid.  Bd.  xxxi.  (1885),  and  Landwirth  Jahrb.  Bd.  xiii.  S. 
513.  Rubner,  Zt.  f.  Biol  Bde.  xix.  (1883),  S.  313 ;  xxi.  Sn.  250,  337.  Berthelot  et 
Andre,  Compt.  Rend.,  T.  ex.  (1890),  p.  884. 

'^  Langley,   Jl.  of  Physiol  Vol.  x.  (1889),  p.  433. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.  77 

appears  that  the  substance  at  first  secreted  by  the  mucous  cells 
(of  Helix)  may  not  be  typical  mucin,  but  a  sort  of  mucinogen 
which  readily  gives  rise  to  mucin  on  treatment  with  dilute 
(•01  p.c.)  caustic  potash.^  If  it  be  assumed  for  the  moment  that 
there  is  only  one  kind  of  mucin,  then  the  following  general  state- 
ments as  to  this  substance  may  be  additionally  made.  It  is  pre- 
cipitated from  its  solutions  by  acetic  or  hydrochloric  acids,  the 
precipitate  being  soluble  in  excess  of  the  latter  but  not  of  the 
former  acid.  In  its  precipitated  form  it  swells  up  strongly  in 
water  but  does  not  go  into  true  solution ;  the  addition  of  dilute 
alkalis  ("1 — "2  p.c.)  or  of  lime-water  leads  to  its  ready  solution, 
from  which  it  can  again  be  precipitated  by  the  addition  of  an 
acid.  It  may  be  extracted  from  any  mucigenous  tissue  by  the 
use  of  dilute  alkalis  or  lime-water,  and  in  solution  is  somewhat 
characteristically  precipitated  by  basic  lead  acetate.  Our  knowl- 
edge of  mucin  is  however  in  an  extremely  transitional  condition, 
and  recent  investigations  have  shown  that  probably  the  mucins 
derived  from  different  sources  are  really  distinct  substances,  just 
as  we  are  familiar  with  different  forms  of  proteids.  From  this  it 
follows  that  no  general  statement  of  the  properties  of  the  mucins 
can  be  as  yet  made  which  would  be  other  than  misleading,  and  it 
will  conduce  to  clearness  to  give  a  brief  account  of  this  substance 
as  obtained  from  each  of  the  chief  sources  from  which  it  has  been 
derived. 

The  mucin  of  hile.^  Mucin  is  not  a  constituent  of  normal  bile 
when  freshly  secreted,  but  is  found  in  it  as  the  result  of  the 
secretory  activity  of  the  internal  epithelivim  of  the  gall-bladder. 
It  is  best  prepared  as  follows  (PaijkuU).  Bile  is  mixed  with 
five  volumes  of  absolute  alcohol  and  centrif  ugalised ;  the  precipi- 
tated mucin  which  is  thus  obtained  is  then  dissolved  in  water 
and  the  above  process  repeated  two  or  three  times.  An  aqueous 
solution  of  this  mucin  is  precipitated  by  acetic  and  hydrochloric 
acids,  is  soluble  in  excess  of  either  acid,  and  yields  strongly 
marked  proteid  reactions.  This  mucin  differs  from  that  obtained 
from  other  sources  in  not  yielding  any  reducing  substance  when 
boiled  with  acids,  and  in  the  solubility  of  its  precipitate  obtained 
by  means  of  acetic  acid  in  an  excess  of  this  acid.  It  also  con- 
tains phosphorus,  and  is  by  some  regarded  as  more  closely  allied 
to  the  nucleo-albumins  (see  p.  89)  than  to  the  true  mucins. 

The  mucin  of  the  sub-maxillary  gland^  The  gland  is  finely 
minced,  washed,  and  extracted  with  water :  the  extract  is  filtered 

1  Hammarsten,  Pfliiger's  Arch.  Bd.  xxxvi.  (1885)  S.  390. 

2  Landwehr,  Zt.  f.  physiol.  Chem.  Bd.  v.  (1881),  S.  371 ;  viii.  (1883),  S.  lU.  Paij- 
kuU, Ibid.  XII.  (1887),  S.  196. 

3  Hammarsten,  Zt.  f.  physiol.  Chem.  xii.  (1888),  S.  163.  Contains  references  to 
other  literature.  Obolensky,  Hoppe-Seyler's  med.-chem.  Unters.  Hfl.  4  (1871),  S.  590. 
Also  in  Pfliiger's  Arch.  Bd.  iv.  (1871),  S.  336. 


78  MUCIN. 

and  hydrochloric  acid  is  added  up  to  1 — 15  p.c.  The  mucin  is 
thus  precipitated  at  first,  but  at  once  passes  into  solution,  from 
which  it  is  precipitated  by  the  addition  of  a  volume  of  water  equal 
to  three  to  five  times  that  of  the  original  solution.  This  precipi- 
tate is  then  again  dissolved  in  dilute  hydrochloric  acid  and  repre- 
cipitated  by  water,  the  process  being  repeated  several  times.  As 
thus  prepared  and  thoroughly  washed  it  possesses  a  distinctly 
acid  reaction ;  it  may  be  dissolved  to  a  neutral  solution  by  the 
cautious  addition  of  mry  dilute  alkalis,  and  now  exhibits  the  fol- 
lowing properties.  It  is  readily  precipitated  by  acetic  acid,  much 
less  readily  in  presence  of  sodium  chloride ;  this  salt  on  the  other 
hand  greatly  facilitates  the  precipitation  of  mucin  by  alcohol,  which 
again  does  not  take  place  in  presence  of  a  trace  of  free  alkali. 
Any  excess  of  alkali,  especially  on  warming,  at  once  changes  the 
substance  so  that  its  characteristic  ropiness  is  permanently  lost, 
and  boiling  with  dilute  mineral  acids  yields  a  reducing  substance. 
It  gives  the  usual  reactions  for  proteids  and  is  strongly  precipi- 
tated by  the  acetates  of  lead  and  by  CuSO*  and  by  excess  of  NaCl 
and  MgS04. 

The  mucin  of  Helix  pomatia}  Hammarsten  distinguishes  be- 
tween the  mucin  contained  in  the  secretion  of  the  mantle  and 
that  which  may  be  derived  from  the  foot  of  this  animal.  Mantle- 
muein.  The  secretion  of  the  mantle  contains  a  mucigenous  sub- 
stance precipitable  by  acetic  acid  which  is  exceedingly  insoluble 
in  water,  but  is  readily  converted  into  true  mucin  by  the  action 
of  dilute  (-01  p.c.)  caustic  potash.  From  its  solution  in  alkali  it 
may  be  purified  by  precipitation  with  acetic  acid,  washing,  re- 
solution in  alkali  and  reprecipitation  with  acid.  When  dissolved 
in  a  trace  of  alkali  the  solution  yields  the  reactions  typical  of 
other  mucins,  but  it  differs  from  these  in  the  fact  that  the  precipi- 
tate formed  on  the  addition  of  hydrochloric  acid  (or  acetic)  is  not 
soluble  in  excess  of  the  acid.  Foot-mucin.  It  may  be  obtained 
by  extracting  the  foot  with  .01  p.c.  KHo;  from  this  solution  it  is 
now  precipitated  by  the  addition  of  hydrochloric  acid  (not  acetic) 
up  to  -1  -  '2  p.c,  redissolved  in  alkali  and  reprecipitated  with  acid, 
the  process  being  repeated  several  times.  Solutions  of  this  mucin 
resemble  those  of  mantle-mucin  in  all  essential  respects,  the  only 
difference  which  is  stated  to  be  characteristic  of  the  two  being  that 
in  presence  of  sodium  chloride,  mantle-mucin,  like  that  of  the 
submaxillary  gland,  is  not  precipitated  by  faint  acidulation  with 
acetic  acid,  whereas  under  similar  conditions  solutions  of  foot- 
mucin  cannot  even  be  neutralised  without  yielding  an  opalescence 
or  precipitate. 

The  mucin  of  tendons.^     The  tendo  Achillis  of  the  ox  is  cut  into 

1  Hammarsten,  Pfluger's  Arch.  Bd.  xxxvi.  (1885),  S.  373.  Gives  previous 
literature. 

2  Lobisch,  Zt.  f.  physiol.  Chem.  Bd.  x.  (1886),  S.  40.     Gives  previous  literature. 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.  79 

thin  slices,  washed  with  distilled  water  and  extracted  with  half- 
saturated  lime-water ;  the  mucin  is  thus  dissolved,  and  is  purified 
by  precipitation  with  either  acetic  or  hydrochloric  acids,  re-solu- 
tion in  dilute  alkali,  and  reprecipitation  with  acids.  In  its  general 
reactions  it  resembles  the  mucins  previously  described,  but  appears 
to  differ  from  them  in  its  distinctly  greater  resistance  to  the  action 
of  acids  and  alkalis. 

Mucin  of  the  umbilical  coi^d}  May  be  extracted  by  means  of 
water  and  is  readily  precipitated  from  the  solution  by  acetic  acid. 
It  appears  to  differ  from  the  other  mucins  in  containing  more 
nitrogen  and  a  considerable  amount  of  sulphur:  it  lies  in  fact 
somewhat  midway  between  the  proteids  and  true  mucins. 

By  prolonged  boiling  with  sulphuric  acid  mucins  yield  leucin 
and  tyrosin,  but  the  products  of  their  decomposition  have  not  been 
as  yet  fully  studied.^ 

Analyses  of  the  several  mucins  exhibit  differences  in  percentage 
composition  which  lie  within  somewhat  similar  limits  to  those  al- 
ready assigned  (p.  5)  to  the  proteids.  A  comparison  of  these  seems  to 
justify  the  statement  that  on  the  whole  the  mucins  contain  slightly 
less  carbon  and  distinctly  less  nitrogen  than  do  the  proteids.^ 

During  his  researches  on  mucin  Landwehr  *  obtained  a  substance  to 
which  he  gave  the  name  of  "animal-gum  "  from  its  general  similar- 
ity to  the  vegetable  products  of  the  same  name.  He  was  at  first  in- 
clined to  regard  the  mucins  as  mixtures  of  this  carbohydrate  with 
other  proteid  substances,  but  this  view  he  subsequently  modified.^ 
Further  investigation  has  led  him  to  regard  animal-gum  as  occurring 
in  many  tissues  of  the  body,  and  to  speculate  on  its  physiological  and 
pathological  significance.^  Its  isolation  from  the  several  tissues  is 
somewhat  lengthy  and  complicated,  and  for  this  Landwehr's  original 
papers  must  be  consulted.  It  dissolves  in  water  to  form  a  readily 
foaming  solution,  from  which  it  may  be  precipitated  by  alcohol.  In 
alkaline  solution  it  readily  dissolves  cupric  oxide  which  is  not  reduced 
on  boiling:  when  boiled  with  dilute  mineral  acids  it  yields  a  reducing 
sugar,  but  it  is  not  altered  by  digestion  with  saliva  or  pancreatic 
juice   (see  also  below  under  carbohydrates). 

It  has  been  already  stated  that  purified  mucin  (except  of  bile) 
yields  a  carbohydrate  when  heated  with  acids  or  stronger  alkalis,  and 
a  considerable  controversy  has  been  carried  on  as  to  whether  animal- 
gum  is  a  carbohydrate  which  occurs  in  the  tissues  as  a  mere  com- 
panion of  the  mucins  or  whether  it  is  in  all  cases  a  product  of  their 
decomposition.  The  evidence  at  hand  on  this  point  is  not  conclu- 
sive, and  for  the  present  it  may  be  said  that,  while  mucin  is  often 

1  Jernstrora  (Swedish).     See  Abst.  in  Maly's  Bericht.  1880,  S.  34. 

2  Walchli,  Jn.  /.  prakt.  Chem.  N.  F.  Bd.  xvii.  (1878),  S.  71. 

3  See  Liebermann,  Biol.  Centralb.  Bd.  vii.  (1887-88),  S.  60. 

*  Zt.  f.  physiol.  Chem.  Bd.  vi.  (1881),  S.  75;  viii.  (1883),  S.  122. 

5  Ibi'd.  Bd.  IX.  S.  367. 

6  Centralb.  f.  d.  med.  Wiss.  (1885),  S.  369.  Pfliiger's  Arch.  Bde.  xxxix.  (1886). 
S.  193;  XL.  S."21. 


so  GELATIN. 

accompanied  by  animal-gum,  the  latter  has  by  no  means  been  proved 
to  take  its  origin  from  the  former.  The  whole  subject  requires 
further  investigation. 

Gelatin  or  Glutin.^ 

The  ultimate  fibrils  of  connective  tissue  and  the  organic  matter 
of  which  bones  are  largely  composed  consist  of  a  substance  named 
in  the  first  case  '  collagen,'  in  the  second  '  ossein.'  They  are  ob- 
tained either  by  digesting  carefully  cleansed  tendons  with  trypsin, 
which  dissolves  up  all  the  tissue-elements  except  the  true  collage- 
nous (gelatiniferous)  fibrils,^  or  by  extracting  bones  with  dilute 
acids  in  the  cold,  by  means  of  which  the  inorganic  salts  are  dis- 
solved and  the  ossein  remains  as  a  swollen  elastic  mass  which  re- 
tains the  shape  of  the  original  bone.  As  thus  prepared  they  are 
insoluble  in  water,  saline  solutions,  and  either  cold  dilute  acids  or 
alkalis ;  in  the  former,  however,  (acids)  they  swell  up  to  a  trans- 
parent gelatinous  mass.  When  subjected  to  prolonged  boiling 
with  water,  more  especially  under  pressure  as  in  a  Papin's  diges- 
ter, they  are  gradually  dissolved,  and  the  solution  now  contains 
true  gelatin  into  which  they  have  been  converted  by  hydrolysis, 
and  has  acquired  the  characteristic  property  of  solidifying  into  a 
jelly  on  cooling.  The  conversion  of  collagen  into  gelatin  may  be 
still  more  easily  effected  by  a  shorter  boiling  in  presence  of  dilute 
acids,  but  in  this  case,  unless  the  process  be  carefully  regulated, 
the  first-formed  gelatin  is  further  hydrolysed  into  what  are  often 
spoken  of  as  gelatin-peptones.  Although  insoluble  in  dilute  acids 
collagen  is  readily  dissolved  by  digestion  with  pepsin  in  presence 
of  an  acid  passing  rapidly  through  the  condition  of  gelatin  into 
that  of  gelatin-peptone,  and  although  collagen  is  not  acted  upon 
by  trypsin  in  alkaline  solution,  it  is  readily  hydrolysed  by  this 
enzyme  after  a  short  preliminary  treatment  with  dilute  acid  or 
boiling  water,  the  products  as  before  being  known  as  gelatin-pep- 
tones. When  gelatin  is  exposed  for  some  time  in  the  dry  condi- 
tion to  a  temperature  of  130°  it  is  reconverted  into  a  substance 
closely  resembling  collagen,  which  may  be  again  converted  into 
gelatin  by  treatment  with  water  under  pressure  at  120°.^ 

Gelatin  obtained  by  the  above  means  from  connective  tissue  or 
bones  is,  when  dry,  a  transparent,  more  or  less  coloured  and  brittle 
substance.*  It  is  insoluble  in  cold  water,  but  swells  up  into  an 
elastic  fl.exible  mass  which  now  dissolves  readily  in  water  when 
warmed.     When  the  solution  is  again  cooled  it  solidities  charac- 

1  Glutiu  must  not  be  confounded  with  the  vegetable  proteid  '  gluten.' 

2  Kiihne  u.  Ewald,  Verhand.  d.  naturhist.-med.  Ver.  Heidelb.  Bd.  i.  N.F.  (1877), 
S.  3.  See  also  Etzinger,  Zt.f.  Biol.  Bd.  x.  (1874),  S.  84.  Ewald,  Ibid.  Bd.  xxvi. 
(1889),  S.  1. 

*  Hofmeister,  Zt.  f.  physiol  Chem.  Bd.  ii.  (1878),  S.  313.  Weiske,  Ibid.  vii. 
(•1883),  S.  460. 

*  Pure  gelatin  is  colourless,  e.  g.  fine  isinglass  prepared  from  the  bladder  of  the 
sturgeon.     Glue  is  impure  gelatin  made  from  hides,  &c. 


CHEMICAL  BASIS  OF  THE  ANIMAL   BODY.  81 

teristically  into  a  jelly  even  when  it  contains  as  little  as  1  p.  c.  of 
gelatin ;  it  is  also  readily  soluble  in  the  cold  in  dilute  acids  and 
alkalis.  The  proteid  reactions  of  gelatin  are  so  feeble  that  they 
must  be  regarded  as  due  entirely  to  unavoidably  admixed  traces  of 
proteid  impurities ;  more  particularly  is  it  to  be  noticed  that  the 
usual  reaction  of  proteids  with  Millon's  reagent  is  entirely  want- 
ing, a  fact  which  indicates  the  probable  absence  of  aromatic  (ben- 
zol) residues  in  its  molecule  and  corresponds  to  the  absence  of 
tyrosin  among  the  products  of  its  decomposition.  Notwithstand- 
ing that  it  is  in  no  sense  a  proteid,  its  percentage  composition  ap- 
proximates to  that  of  the  latter  class  of  substances  and  may  be 
taken  as  C  =  50-76,  H  =  7-15,  0  =  23-21,  N  =  18-32,  from  which 
it  appears  to  contain  distinctly  less  carbon  than  do  the  proteids ; 
it  is  also  stated  to  contain  no  sulphur  when  pure,  but  ordinarily  it 
contains  a  small  amount  (-5  p.  c.).^  Gelatin  is  precipitated  by  but 
few  salts,  viz. :  mercuric  chloride  and  the  double  iodide  of  mer- 
cury and  potassium  in  acid  solution.  Several  acids  on  the  other 
hand  precipitate  it  readily,  such  as  phosphotungstic  and  meta- 
phosphoric,  also  taurocholic  and  tannic.  Of  the  two  last-named 
acids  the  former  yields  an  opalescence  in  presence  of  1  part  of 
gelatin  in  300,000  of  solution,  and  the  latter  in  still  more  dilute 
solutions."^  The  precipitability  with  tannic  acid  seems  to  depend 
on  the  presence  of  neutral  salts.^  The  specific  rotatory  power  of 
gelatin  in  aqueous  solution  or  in  presence  of  a  trace  of  alkali  is 
stated  to  be  {a)^==-ldO°  at  30°  C.  and  to  be  reduced  to  -112° 
or  -114°  on  the  addition  of  more  alkali  or  acetic  acid.*  This 
statement  requires  confirming. 

When  decomposed  in  seal  tubes  with  caustic-baryta  gelatin 
yields  on  the  whole  the  same  products  as  do  the  proteids,^  with 
the  exception  of  tyrosin ;  neither  this  nor  any  other  substance  of 
the  typically  aromatic  series  is  ever  obtained  during  any  decom- 
position of  gelatin,  whether  by  chemical  or  putrefactive  processes.^ 
By  prolonged  boiling  with  hydrochloric  acid  it  yields  glycin 
(glycocoll),  leucin,  glutamic  acid,  and  ammonia,"  and  with  sul- 
phuric acid  aspartic  acid  as  well.^ 

Gelatin-peptones.^  By  prolonged  boiling  with  water  (1  p.c.  so- 
lution boiled  for  30  hours),  or  shorter  treatment  in  aPapin's  diges- 

1  Hammarsten  Zt.  f.  phi/siol  Chem.  Bd.  ix.  (1885),  S.  305. 

2  Emich  Monatshejief.  Chem.  Bd.  vi.  (1885),  S.  95. 
^  Weiske,  loc.  cit. 

*  J.  de  Bary,  Diss.  Tubingen,  1864.  Also  in  Hoppe-Seyler's  med.-chem.  Unters. 
Bit.  1,  1866,  S.  73. 

^  Schiitzenberger  et  Bourgeois,  Compt.  Rend.  T.  lxxxii.  (1876),  p.  262. 

6  Nencki.  See  Abst.  in  Maly's  Bericht.  1876,  S.  31.  Jeanneret,  Jn.  f.  prakt. 
Chem.  (N.F.)  Bd.  xv.  (1877),  S.  353.  Wevl,  Zf.  f.  physiol.  Chem.  Bd.  i.  (1877),  S. 
339. 

■^  Horbaczewski,  Sitzb.  d.  Wien.  Ahad.  Bd.  lxxx.  (1879),  2  Abth.  Juni.-Hft. 

8  Gaehtgens,   Zt.f.  physiol.  Chem.  Bd.  i.  (1877),  S.  299. 

^  Hofmeister,  Zt.f.  physiol.  Chem.  Bd.  ii.  (1878),  S.  299.  Gives  literature  down 
to  that  date.     Tatarinoff, 'Co?np<.  iienrf.  T.  xcvii.  (1883),  p.  713. 

6 


82  GELATIN. 

ter,  also  by  heating  with  hydrochloric  acid  (4  p.c.  at  40°),  or  still 
more  readily  by  pepsin  in  presence  of  acid  or  by  trypsin,^  gelatin 
loses  its  power  of  solidifying  on  cooling,  and  is  converted  into 
more  highly  soluble  and  now  diffusible  substances,  to  which  the 
name  of  gelatin-peptones  has  been  given.  A  similar  change 
occurs  when  gelatin  is  taken  into  the  stomach. ^  From  the  con- 
ditions under  which  the  change  is  effected  and  from  certain  evi- 
dence deducible  from  analysis  there  can  be  but  little  doubt  that 
the  conversion  takes  place  as  the  result  of  hydrolysis,  as  in  the 
case  of  the  formation  of  true  peptones  from  proteids. 

Eecent  researches  have  shown  that  the  hydrolytic  decomposi- 
tion of  gelatin  by  digestive  enzymes  gives  rise  to  products  analo- 
gous to  those  obtainable  by  the  same  method  from  the  proteids. 
Thus  during  both  its  peptic  and  tryptic  digestion  certain  primary 
products  are  formed  to  which  the  name  gelatoses  or  glutoses  may 
be  applied,  and  which  have  so  far  been  distinguished  as  proto-  and 
deutero-gelatose.  Accompanying  these,  in  variable  amount,  are 
other  products  known  as  gelatin-peptones.  The  latter  are  to  be 
regarded  as  a  product  of  the  further  action  of  the  enzymes  on  the 
first  formed  gelatoses  and,  like  the  true  peptones  in  their  relation- 
ship to  the  albumoses,  may  be  separated  from  them  by  their  non- 
precipitability  on  saturation  with  ammonium  sulphate,  a  reagent 
which  completely  precipitates  the  gelatoses.  Protogelatose  is 
partially  precipitated  by  saturation  of  its  solution  with  common 
salt,  and  completely  so  on  the  simultaneous  addition  of  acetic 
acid.  Deuterogelatose  is  not  precipitated  by  either  of  the  above 
reagents.^  The  so-called  true  gelatin-peptones  have  not  yet  been 
obtained  in  sufficient  quantity  to  admit  of  their  complete  exami- 
nation. The  products  of  the  digestion  of  gelatin  appear  to  give 
a  distinct  biuret  reaction  with  caustic  soda  and  sulphate  of  cop- 
per, and  like  the  peptones  (and  albumoses)  are  not  precipitated 
by  taurocholic  acid,  which  precipitates  gelatin  from  its  solutions.* 

When  the  spores  of  PeBicillium  are  sown  on  a  surface  of  gelatin,  as 
soon  as  the  mycelium  is  well  developed  the  subjacent  gelatin  liquefies 
sometimes  to  a  considerable  depth,  so  that  the  Penicillium  finally 
floats  on  a  layer  of  fluid  separated  by  some  distance  from  the  re- 
maining still  solid  gelatin.  The  fluid  in  this  laj^er  now  yields  an 
intense  biuret  reaction.  A  similar  liquefaction  is  observed  during 
the  growth  of  certain  bacteria  and  other  micro-organisms  on  gelatin. 

The  fact  has  already  been  referred  to  (§  524)  that  gelatin  taken  as 
food,  while  it  materially  lessens  both  the  nitrogenous,   and  to  some 

1  Schweder,  Inauq.-Diss.  Berlin,  1867. 

2  Uffelmann,  Arch.  f.  Uin.  Med.  Bd.  xx.  (1877),  S.  535. 

3  Chittenden  and  Solley,  Jl.  of  Physiol.  Vol.  xii.  (1891),  p.  23.  See  also  Klug, 
Pfliiger's  Arch.  Bd.  xlviii.  (1890),  S.  100.  The  latter  author  describes  further  a 
product  to  which  he  gives  the  name  apoglutin.  It  makes  its  appearance  as  an 
insoluble  substance,  hence  resemhling  antialbumid  or  dyspeptone,  during  the  diges- 
tion of  gelatin. 

4  Emich,   Monatshefle  f.  Chem.  Bd.  vi.  (1885),  S.  95. 


CHEMICAL   BASIS    OF   THE   ANIMAL   B0I3Y.  S'S 

slight  extent  the  non-nitrogenous  metabolism  of  the  body,  and  thus 
appears  able  to  undergo  a  destructive  metabolism  similar  to  that  of 
the  proteids,  cannot,  on  the  other  hand,  play  any  part  in  the  con- 
structive nitrogenous  metabolism  which  leads  to  the  formation  of  pro- 
teids. In  other  words  the  nitrogen  contained  in  gelatin  cannot  be 
built  up  into  the  nitrogen  of  a  proteid.^  We  do  not  as  yet  possess 
any  information  which  enables  us  to  formulate  any  reason  for  this 
special  behaviour  of  gelatin.  It  has  been  suggested  that  the  absence 
of  aromatic  residues  in  gelatin  (see  above)  might  account  for  the  phe- 
nomenon,^ but  experiments  in  which  animals  have  been  fed  with 
gelatin-|-tyrosin  have  not  confirmed  this  view.^  It  appears  that  g, 
considerable  amount  of  gelatin  is  digested  and  absorbed  in  man,  since 
none  appears  in  the  faeces,  and  meat  (muscle)  may  contain  as  much 
as  2  p.  c.  of  gelatin :  further,  Voit's  experiments  show  that  a  dog  may 
digest  and  absorb  50  p.c.  of  the  gelatin  administered  in  the  form  of 
bones.*  Bearing  these  facts  in  mind  and  knowing  that  gelatin  ap- 
pears to  be  more  readily  metabolised  than  proteids,  we  may  regard 
gelatin  as  a  valuable  food-stuff,  but  not  as  a  food  which  can  sujjply 
the  nitrogenous  needs  of  the  tissues  themselves.  The  facts  thus 
stated  may  supply  an  explanation  of  the  beneficial  effects  which  are 
supposed  to  result  from  the  use  of  jellies  in  training  diets. ^ 

Chondrin. 

The  matrix  of  hyaline  cartilage  is  composed  of  an  elastic,  semi- 
transparent  substance  which  is  insoluble  in  cold  or  hot  water  and 
does  not  swell  up  appreciably  by  treatment  with  either  water  or 
dilute  acetic  acid.  By  prolonged  treatment  with  water  under 
pressure  in  a  Papin's  digester  it  is  gradually  dissolved  and  yields 
a  solution  which  gelatinises  on  cooling  and  now  contains  the 
substance  usually  spoken  of  as  chondrin.  The  hyaline  matrix  of 
cartilage  appears  thus  to  bear  the  same  relationship  to  chondrin 
that  the  ground-substance  of  connective-tissue  (collagen)  does  to 
gelatin,  and  is  therefore  frequently  spoken  of  as  '  chondrigen.' 

The  substance  known  as  chondrin,  which  is  obtained  in  solution 
by  the  action  of  superheated  water  on  hyaline  cartilage,  exhibits 
the  following  characteristic  reactions.^  It  is  precipitated  by  acetic 
acid,  which  does  not,  even  if  in  considerable  excess,  redissolve  the 
precipitate ;  minute  quantities  of  mineral  acids  similarly  cause  a 
precipitate  to  appear  which  is  in  this  case  readily  soluble  in  the 
slightest  excess  of  the  acids.  These  reactions  suffice  to  distinguish 
between  chondrin  and  gelatin,  and  a  further  distinction  may  be 
made  on  the  basis  of  the  fact  that  solutions  of  chondrin  are  -pre- 
cipitated  by  several  reagents  such  as  alum,  normal  lead  acetate, 

1  Voit,  Zt.f.  Biol.  Bd.  viii.  (1872),  S.  297  ;  x.  (1874),  S.  203. 

-  Hermann  u.  Escher,  Viei-teljahrxch.  d.  natforsch.  Gesell.  in  Zurich,  1876,  S.  36. 

3  Lehmann,  Sitzber.  d.  Gesell.  f.  Morphol.  u.  Physiol.  Miinchen,  1885. 

4  See  also  Etzinger,   Zt.f.  Biol.  Bd.  x.  (1874),  S.  84. 

^  For  a  statement  of  the  nutritional,  metabolic  and  physiological  significance  of 
gelatin  see  Hermann's  Hdbch.  d.  Physiol.  Bd.  vi.  Sn.  123,  318,  391,  395. 
6  Moleschott  u.  Fubini,  Moleschott's  Untersuch.  Bd.  xi,  (1872),  S.  104. 


84  CHONDKIK 

and  other  metallic  salts  (of  Ag  and  Cu),  which  yield  no  precipitate 
with  gelatin,  while  on  the  other  hand  mercuric  chloride  and  tan- 
nin do  not  precipitate  chondrin  but  are  characteristic  precipitants 
of  gelatin  (see  above).  Chondrin  is  powerfully  laevorotatory ;  in 
faintly  alkaline  solution  (a)j)  =  -  213'5°  ;  in  presence  of  excess  of 
alkali  this  becomes  (a)j)  =  -  55-20°.i 

By  prolonged  treatment  with  boiling  water,  or  shorter  heating 
with  dilute  (1  p.c.)  sulphuric  acid  or  stronger  hydrochloric  acid, 
chondrin  is  decomposed  with  the  formation  of  a  nitrogenous  crys- 
tallisable  product  which  characteristically  reduces  alkaline  solu- 
tions of  cupric  oxide.^  Opinions  however  differ  considerably  as  to 
the  real  nature  of  this  reducing  substance.  It  was  at  one  time 
regarded  as  a  true  carbohydrate,  and  more  recently  Landwehr  has 
identified  it  with  his  animal-gum.^  (See  above  suh  mucin.)  There 
is  now  but  little  doubt  that  it  contains  nitrogen,  is  possessed  of 
distinct  acid  properties,  and  exhibits  marked  carbohydrate  affini- 
ties apart  from  its  reducing  powers.*  According  to  the  older  and 
some  recent  observers  its  solutions  are  laevorotatory,^  but  v,  Mer- 
ing  states  that  it  is  dextrorotatory.^  Its  real  nature  cannot  be 
regarded  as  yet  as  definitely  established.  When  the  action  of  the 
boiling  acids  is  prolonged,  or  if  caustic  alkalis  or  barium  hydrate 
is  employed,  chondrin  undergoes  a  further  profound  decomposition 
resulting  in  the  formation  of  a  large  number  of  crystalline  pro- 
ducts ;  with  regard  to  these  the  fact  of  chief  importance  and 
interest  is  the  general  presence  among  them  of  leucin,  and  the 
entire  absence  of  tyrosin  and  glycin  (glycocoU),  and  the  occurrence 
of  aspartic  and  glutamic  acids  in  very  minute  traces  only,  if  at  all.'' 

We  have  so  far  spoken  of  chondrin  as  a  distinct  and  individual  sub- 
stance; the  view  has  however  been  put  forward  that  it  is  in  reality 
merely  a  mixture  of  mucin  and  gelatin,^  and  the  outcome  of  more  re- 
cent work  seems  to  be  tending  towards  the  strengthening  of  this  view.® 
When  hyaline  cartilage  is  extracted  with  baryta  water  or  dilute  alkalis 
a  solution  is  obtained  which  yields  reactions  tj'^pical  of  the  so-called 
chondrin  and  closely  resembling  those  characteristic  of  mucin;  the 
undissolved  residue  when  boiled  with  water  is  dissolved  into  a  solution 
which  gives  the  reactions  in  general  typical  of  gelatin.  Morner, 
treating  sections  of  hyaline  cartilage  in  succession  with  dilute  hydro- 
chloric acid  (4 — -2  p.c.)  and  caustic  potash  ('1  p.c),  finds  that 
rounded  masses  of  the  matrix  are  dissolved  out  and  leave  thu.i  a  resid- 

1  de  Bary,  loc  cit.  (sub  gelatin). 

2  V.  Mering,  Inauff.-Diss.  Strassburg,  1873. 

3  Pfluger's  Arch.  Bd.  xxxix   (1886),  S.  198. 

*  Krukenberg,  Zt.  f.  BioL.  Bd.  xx.  (1884),  S.  307.  Morner  (Swedish).  See  abst. 
in  Maly's  Bericht.  1887,  S.  308,  1888,  S.  217. 

^  Petri,  Bei-.  d.  deutsch.  chem.  Gesell.  Jahrg.  xii.  (1879),  S.  267. 

•>  See  Hoppe-Seyler's  Hdbch.  d.  physwl.-path.  chem.  Anal.  (5  Auf.  1883),  S.  301. 

■^  Schiitzenberger  et  Bourgeois,  cit.  (sub  gelatin). 

**  Morochowetz,  Verhand.  d.  naturhist.-mea.  Ver.  Heidelbg.  Bd.  i.  (1876),  Hft.-5. 

^  Krukenberg,  Morner,  loc.  cit. 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.  85 

ual  network.  The  dissolved  parts  consist  largely  of  a  substance  (clion- 
dromucoid)  with  marked  affinities  to  mucin,  whereas  the  undissolved 
network,  by  treatment  with  acids  or  superheated  water,  is  converted 
largely  into  typical  gelatin.  For  further  details  the  original  papers 
already  quoted  should  be  consulted. 

Elastin. 

This  is  the  characteristic  component  of  the  elastic  fibres  which 
remain  after  the  removal  of  gelatin,  mucin,  fats,  etc.,  from  tissues 
such  as  "  ligamentum  nuchae."  Some  of  the  more  important  ways 
in  which  it  differs  from  the  substances  which  have  been  previously 
described  are  sufficiently  stated  by  describing  the  method  of  its 
preparation  in  a  pure  form.^  Ligamentum  nuchae  of  an  ox  is  cut 
into  fine  slices,  treated  for  three  or  four  days  with  boiling  water, 
then  for  some  hours  with  1  p.c.  caustic  potash  at  100°C  and  subse- 
quently with  water.  This  process  is  then  repeated  with  10  p.c. 
acetic  acid.  Finally  it  is  treated  for  24  hours  in  the  cold  with  5 
p.c.  hydrochloric  acid,  washed  with  water,  boiled  with  95  p.c.  alco- 
hol, and  extracted  for  at  least  two  weeks  with  ether  to  remove 
every  trace  of  adherent  fat.  By  the  above  method  it  may  be  ob- 
tained as  a  pale  yellowish  powder  in  which  the  shape  of  fragments 
of  the  original  elastic  fibres  may  be  still  distinguished  under  the 
microscope.  When  moist  it  is  yellow  and  elastic,  but  on  drying 
it  becomes  brittle  and  may  with  difficulty  be  pulverised  in  a  mor- 
tar. Sulphur  probably  does  not  enter  into  its  composition  (?).  It 
may  be  dissolved  by  strong  alkalis  at  100°C,  and  it  also  goes  into 
solution  when  treated  with  mineral  acids  at  the  same  temperature  ; 
but  in  the  latter  case  the  solution  involves  decomposition  with  the 
formation  of  much  leucin  (30 — 40  p.c.)  and  traces  (-25  p.c.)  of 
tyrosin  when  the  acid  employed  is  sulphuric. ^  If  strong  hydro- 
chloric acid  be  employed  with  chloride  of  zinc  the  same  crystalline 
products  are  obtained  together  with  ammonia,  glycocoll,  and  an 
amidovalerianic  acid,  but  no  glutamic  or  aspartic  acids.^  In  this 
respect  it  differs  from  both  ordinary  proteids  and  gelatin,  since  the 
former  when  similarly  treated  yield  the  glutamic  and  aspartic 
acids  but  no  glycocoll,  and  the  latter  never  yields  the  least  trace 
of  tyrosin.  During  the  putrefactive  decomposition  of  elastin  pro- 
ducts similar  to  the  above  are  obtained  together  with  some  pep- 
tone-like substance.^  When  treated  with  superheated  water,  or 
with  dilute  hydrochloric  acid  at  100° C.  or  with  pepsin  or  trypsin 
in  acid  and  alkaline  medium  respectively,  elastin  is  more  or  less 
rapidly  dissolved  and  undergoes  a  true  digestive  change,  during 
which  products  are  formed  many  of  whose  general  reactions  are 

1  Horbaczewski,  Zt.  f.  physiol.  Chem.  Bd,  vi.  (1882),  S.  330.  Chittenden  and 
Hart,  Zt.f.  Biol.  Bd.  xxv.  (1889),  S.  368. 

2  Erlenmeyer  u.  Schoffer,  Jn  f.  prakt.  Chem.  Bd.  lxxx.  (1860),  S.  357. 

3  Horbaczewski,  Monutshefie  f.  Chem.  Bd.  vi.  (1885),  S.  639 

*  Walchli,  Jn.  f.  prakt.  Chem.  (N.F.),  Bd.  xvii.  (1878),  S   71. 


86  ELASTIC.     KEEATIN. 

analogous  to  those  of  the  digestive  products  of  proteids.^  It  is 
however  as  yet  uncertain  whether  a  true  elastinpeptone  can  be 
obtained ;  it  is  more  probable  that  during  the  digestion  only  some 
of  the  primary  substances  (elastoses)  make  their  appearance,  since 
they  are  completely  precipitated  by  saturation  with  neutral  am- 
monium sulphate.^.  Elastin  is  also  rapidly  corroded  and  dissolved 
by  the  action  of  papain.     (Gamgee.) 

Hilger  ^  has  obtained  a  somewhat  similar  substance  from  the 
shell  and  yolk  of  certain  snakes'  eggs. 

Keratin. 

Hair,  nails,  feathers,  horn,  and  the  epidermal  structures  in  gen- 
eral are  composed  chiefly  of  keratin,  admixed  however  with  small 
quantities  of  proteids  and  other  substances,  from,  which  it  may  be 
freed  by  thorough  extraction  with  water,  alcohol,  ether,  and  dilute 
acids  in  succession,  followed  by  digestion  with  pepsin  and  trypsin 
(Klihne)  and  a  renewed  washing  with  the  above  reagents.  A  con-' 
venient  source  which  readily  yields  a  pure  product,  owing  to  the 
comparatively  simple  composition  of  the  mother  substance,  is 
found  in  the  shell-membrane  of  ordinary  eggs.*  The  percentage 
composition  of  keratin  is  in  general  allied  to  that  of  the  true  pro- 
teids, but  varies  within  somewhat  wide  limits  according  to  the 
source  from  which  it  has  been  prepared  and  particularly  with  re- 
gard to  the  sulphur  which  it  contains  This  latter  element  varies 
in  amount  from  -5  to  5-0  p.c.  and  leads  to  the  formation  of  sul- 
phides of  the  metal  when  keratin  is  dissolved  in  alkalis.  Unlike 
the  proteids,  gelatin  and  elastin,  keratin  is  quite  unaffected  by  the 
most  prolonged  and  active  digestion  with  either  pepsin  or  trypsin. 
On  the  other  hand,  when  decomposed  at  high  temperatures  by 
either  caustic  baryta  or  strong  hydrochloric  acid,  it  yields  large 
quantities  of  leucin  (15  p.c),  tyrosin  (3 — 4  p.c.)  and  other  pro- 
ducts which  are  in  general  identical  with  those  obtained  by  the 
similar  treatment  of  proteids.^  It  is  soluble  in  strong  alkalis  when 
heated,  and  is  further  stated  to  be  dissolved  by  prolonged  treat- 
ment with  superheated  water ;  in  the  latter  case  a  product  is  ob- 
tained to  which,  since  it  somewhat  resembles  an  albumose,  the 
name  keratinose  has  been  given,  and  which  may  now  be  digested 
by  means  of  pepsin.^  Further  investigation  in  this  direction  is 
however  needed  before  any  positive  statements  can  be  made  re- 
specting any  truly  digestive  products  derivable  from  keratin,  or 
indeed  as  to  the  characteristic  differences  of  the  keratins  from 
different  sources. 

1  Horbaczewski,  loc.  cit. 

2  Chittenden  and  Hart,  loc.  cit. 

3  Ber.  d.  deutsch.  chem.  Gesell.  1873,  S.  166.  See  also  Krukenberg,  Vergl.-physiol. 
Stud.  II.  R.  1.  Abth.  S.  68. 

■*  Lindwall  (Swedish).     See  abst.  in  Maly's  .Tahresber.  1881,  S.  38. 
5  Horbaczewski,  Sitzb.  d.  k.  Akad.  d.  Wiss.  Wien.  Bd.  i.xxx.    (1879),  2  Abth. 
Juni-Hft.     Bleunard,  Compt.  Rend.  T.  Lxxxix.  (1879),  p  953,  T  xc.  (1880),  p.  612. 
s  Krukenberg,  Sitzb.  d.  Jena,  Gesell.  f.  Med.  u.  Nat.-iviss.  1886,  S.  22. 


CHEMICAL   BASIS   OF   THE   ANIMAL   BODY.         87 

Lindwall  (loc.  eit.)  described  the  formation  of  an  albuminate  and  a 
peptone-like  (?  albumose)  substance  during  the  treatment  of  keratin 
with  dilute  (1  —  2  p.c.)  caustic  soda  at  digestion  temperatures. 

Neurokeratin.^ 

When  the  substance  of  the  brain  or  any  mass  of  medullated 
nerves  is  thoroughly  extracted  with  water,  alcohol,  and  ether,  and 
then  digested  with  pepsin  and  trypsin  in  succession,  a  residue  is 
obtained  which  closely  resembles  the  ordinary  keratins,  and  con- 
stitutes about  15 — 20  p.c.  of  the  whole  brain  after  it  has  been 
freed  from  its  medullary  constituents  by  alcohol  and  ether.^  This 
residue  is  neurokeratin,  so  named  from  the  source  from  which  it 
is  obtained.  It  is  characterised  by  its  somewhat  greater  resistance 
to  those  decomposing  agents  whose  action  on  keratin  has  been 
already  described.  The  determination  of  its  existence  in  tissues 
which  are  not  obviously  epidermal  in  the  adult  is  of  considerable 
embryological  and  morphological  interest,  since  it  throws  some 
light  upon  the  developmental  origin  of  the  structures  in  which  it 
is  present  or  absent.^ 

Chitin.    C15H26N2O10.' 

Although  it  is  not  found  as  a  constituent  of  any  mammalian 
tissue,  this  substance  composes  the  chief  part  of  the  exoskeleton 
of  many  invertebrates.  It  is  by  many  regarded  as  the  animal 
analogue  of  cellulose  of  plants,  and  from  this  point  of  view  it 
possesses  considerable  morphological  interest.  The  most  con- 
venient source  from  which  it  may  be  prepared  is  the  cleansed 
exoskeleton  of  crabs  or  lobsters.  This  is  first  thoroughly  extracted 
with  dilute  hydrochloric  acid  and  caustic  potash,  after  which  it  is 
treated  with  boiling  alcohol  and  ether,  and  may  be  finally  com- 
pletely decolorised  by  the  action  of  permanganate  of  potash.^  It 
is  a  white  amorphous  substance,  which  often  retains  the  shape  of 
the  integument  from  which  it  has  been  prepared.  It  is  insoluble 
in  any  reagents  other  than  concentrated  mineral  acids,  such  as 
sulphuric  or  hydrochloric.  The  immediate  addition  of  water  to 
these  solutions  probably  reprecipitates  the  chitin  in  an  unaltered 
form.^  When  heated  with  concentrated  hydrochloric  acid  it  is 
decomposed  into  glycosamin  and  acetic  acid,  of  which  the  former 

1  Kiihne  u  Ewald,  Ve.rhand,  naturhist.-med.  Ver.  Heidelbg.  Bd.  1,  1877,  S.  457. 
Kiihne  u.  Chittenden,  Zt.  f.  Biol.  Bd.  xxvi.  (1890),  S.  291. 

2  See  also  Chevalier,  Zt.  f.  vhysiol.  Chem.  Bd.  x.  (1886),  S.  100. 

3  Cf.  Smith,  H.  E.,  Zt.  f.  Biol  Bd.  xix.  (1883),  S.  469.  Steiubriigge,  Ibid.  Bd. 
XXI.' (1885),  S.  631. 

*  Ledderhose,  Zt.  f.  physiol  Chem.  Bd.  ii.  (1878),  S.  213.  But  see  also  Suudwik, 
Ihd.  Bd.  V.  (1881),  S.  384. 

5  Biitschli,  Arch.  f.  Anat.  u.  Physiol.  Jahrg.  1874,  S.  362. 

6  But  see  Hoppe-Seyler,  Hdbch.  d.  physiol. -path.  Anal.  5  Aufl.  1883,  S.  188. 
Krukenberg,  Zt.f.  Biol.  Bd,  xxii.  (1886),  S.  480 


88  CHITIN.    NUCLEIK 

is  the  characteristic  product.  ^  A  similar  decomposition  is  observed 
when  sulphuric  acid  is  employed. 

OlycosaTnin  (CeHigNOs).  Crystallises  from  alcohol  in  fine  needles, 
is  dextrorotatory,  and  reduces  Fehling's  fluid  to  the  same  extent  as  does 
dextrose,  but  is  not  fermentable.  By  treatment  with  nitrous  acid  a 
carbohydrate  (CgHiaOe)  (?)  is  obtained  which  also  reduces  cupric 
oxide,  but  is  similarly  unfermentable.  This  is  doubtless  the  sub- 
stance which  led  to  certain  erroneous  statements  as  to  the  production 
of  a  true  dextrose  from  chitin.^ 

Nuclein.     CagH^gNgPsOga  (?). 

The  nuclei  of  cells,  both  animal  and  vegetable,  differ  distinctly 
in  chemical  composition  from  the  remaining  substance  of  the  cells. 
As  a  result  of  this  difference  it  is  possible  to  separate  the  nuclei 
approximately  by  various  means  from  the  adjacent  cell-substance. 
The  name  nuclein  is  given  to  the  material  of  which  the  nuclei  or 
parts  of  nuclei  thus  isolated  chiefly  consist.  When,  however,  the 
statements  of  the  various  authors  who  have  dealt  with  nuclein 
are  compared  with  regard  to  the  reactions,  decompositions,  and 
more  especially  the  percentage  composition  of  their  preparations, 
it  appears  probable  that  no  definite  substance  exists  to  which  the 
one  name  nuclein  may  be  fitly  applied.  It  may  be  that  the  dis- 
crepancies are  due  to  the  existence  of  several  kinds  of  nuclein ;  ^ 
but  this  is  as  yet  scarcely  proved,  and  it  is  on  the  whole  more 
probable  that  the  different  results  of  the  various  authors  must  be 
attributed  to  the  impurity  of  the  substance  on  which  they  op- 
erated.* In  accordance  with  this  view  it  is  to  be  observed  that 
the  percentage  of  phosphorus  obtained  in  even  the  most  reliable 
analyses  is  stated  to  vary  from  2-3  to  9-6  p.c. 

After  the  above  precautionary  remarks  we  may  now  give  an 
account  of  the  preparation  and  properties  of  the  so-called  nuclein. 
"When  a  mass  of  cells  such  as  pus,°  yeast,^  nucleated  red  blood- 
corpuscles,"  salmon-milt,^  or  egg-yolk  ^  is  extracted  with  water  and 
dilute  ('5  p.c.)  hydrochloric  acid,  the  cells  are  largely  broken  up 

1  Ledderhose,  he.  cit.  and  Ibid.  Bd.  iv.  (1880),  S.  139. 

2  Berthelot,  Compt.  Rend.  T.  xlvii.  (1858),  p.  227.  Joiirn.  de  la  Physiol.  T.  ii, 
p.  577. 

3  Hoppe-Seyler,  Hdhch.  d.  ph/jsiol.-path.  cliem.  Anal.  (5  Auf.),  1883,  S.  303. 
Physiol.  Chem.  S.  85. 

*  Worm-Miiller,  Pfluger's  Arch.  Bd.  viii.  (1874),  S.  190.  Bunge,  Physiol. -pathol. 
Chem.  (Transl.  by  Wooldridge,  1890),  p.  89 

s  Miescher,  Hoppe-Seyler's  Med.-chem.  Untersuch.  Hft.  iv.  (1871),  S  452.  Hoppe- 
Sevler,  Ibid.  S.  486. 

'«  Hoppe-Seyler,  Ibid.  S,  500.  Kossel,  Zt.  physiol.  Chem.  Bd.  in.  (1879),  S.  284; 
IV.  (1880),  S.  290;  vii.  (1883),  S.  7.  Unters.  ilb.  d.  Nucleme  u.  ihre  Spaitunqsprod - 
Strassb.  1881.     Loew,  Pfluger's  Arch.  Bd.  xxii.  (1880),  S.  62. 

■?  Bruntou,  Jl.  Anat.  and  Physiol.  2  Ser.  Vol.  in.  1869,  p.  91.  Pldsz,  Hoppe- 
Seyler's  Med.-chem.  Unters.  Hft.  iv.  (1871),  S,  461, 

8  Miescher,  Verhand.  d.  Natforsch.  Gesell.  Basel,  Bd.  vi.  (1874),  S.  138. 

^  Miescher,  Hoppe-Sejler's'^l/ef/.-c^em.  Unters.  Hft.  iv.  (1871),  S.  502.  Worm- 
Miiller,  loc.  at. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  89 

and  dissolved,  and  the  nuclei  separated  from  them.  A  further 
purification  is  obtained  by  treatment  with  alcohol  and  ether  and 
final  digestion  with  pepsin  in  acid  solution,  which  does  not  affect 
the  substance  of  the  nuclei.^  The  final  residue  thus  obtained  is 
washed  with  dilute  acid,  dissolved  in  very  weak  caustic  soda, 
precipitated  by  hydrochloric  acid,  and  washed  with  water  and 
alcohol.  Prepared  by  the  above  methods,  nuclein  is  an  amorphous 
substance,  rich  in  phosphorus,  which  is  set  free  as  phosphoric  acid 
when  it  is  boiled  with  alkalis.  At  the  same  time  some  form  of 
proteid  usually  makes  its  appearance,  as  also  do  the  crystalline 
substances  of  the  xanthin  series,  guanin  (?)  and  hypoxanthin, 
when  the  nuclein  is  heated  with  dilute  mineral  acids  instead  of 
alkalis."^  It  appears,  however,  that  the  absolute  and  relative 
amount  of  the  above  possible  products  of  its  decomposition  varies 
with  the  source  from  which  the  nuclein  is  obtained. 

Under  the  name  '  adenin '  Kossel  has  more  recently  described  a  new 
base  which  he  obtained  by  the  decomposition  of  nuclein  from  yeast-cells 
with  dilute  sulphuric  acid  and  heat.^  It  is  crystalline,  readily  soluble 
in  warm  water  and  caustic  alkalis,  and  when  treated  with  nitrous  acid 
yields  hypoxanthin.      (See  below.) 

CsHsN^+H^O^CsH^K^O+NHs. 

When  egg-  or  serum-albumin  is  precipitated  with  metaphosphoric 
acid,  a  phosphorised  substance  is  obtained  which  exhibits  many  of  the 
reactions  characteristic  of  niiclein.*  It  does  not,  however,  yield  any 
of  the  xanthin  bases  when  treated  with  acids. ^ 

Nucleo-albumins . 

While  the  nuclei  may  be  regarded  as  composed  principally  of 
the  somewhat  unsatisfactorily  characterised  nucleins,  there  is  evi- 
dence of  the  existence  ^  of  closely  allied  substances  to  which,  since 
they  appear  to  be  a  compound  of  nuclein  and  a  proteid,  the  name 
nucleo-albumin  has  been  given.  Our  knowledge  of  these  sub- 
stances is  as  yet  rudimentary  and  imperfect,  and  subsequent 
investigation  must  decide  their  real  nature  and  their  relationship 
to  the  nucleins. 

The  more  characteristic  reactions  of  the  nucleo-albumins  may 
be   stated  as  follows.     Soluble  in  very  dilute  alkalis,  they  are 

1  It  also  resists  the  action  of  trypsin.  Bokay,  Zt.  f.  ph>/siol.  Chem.  Bd.  i.  (1877), 
S.  157. 

2  Kossel,  loc.  cit.  Also  Zt.  f.  physiol.  Chem.  Bd.  v.  (1881),  Sn.  152,  267  ;  Bd.  viii. 
(1884),  S.  404. 

3  Ber.  d.  d.  chem.  Gesell.  1885,  Sn.  79,  1928.  Zt.  physiol.  Chem.  Bd.  x.  (1886), 
S.  250.  Schindler,  Ibid.  Bd.  xiii.  (1889),  S.  432.  Bruhns,  Ibid.  Bd.  xiv.  (1890), 
S.  533. 

4  Liebermann,  Ber.  d.  d.  chem.  Gesell.  (1888),  S.  598.  Pfliiger's  Arch.  Bd.  xlvii. 
(1890),  S.  155. 

5  Pohl,  Zt.  f.  physiol.  Chem.  Bd.  xiii.  (1889),  S.  292. 

6  Worm-lMuller,  Pfliiger's  Arch.  Bd.  viii.  (1874),  S.  194. 


90  NUCLEO-ALBUMmS. 

readily  reprecipitated  by  acetic  acid ;  and  the  constancy  in  prop- 
erties of  the  product  obtained  by  repeated  solution  and  precipita- 
tion seems  to  show  that  they  are  not  mere  mixtures  of  nuclein 
and  proteid.  Their  behaviour  towards  alkalis  and  acetic  acid  is 
such  as  to  lead  to  an  easy  confusion  with  the  mucins.  When 
digested  with  pepsin  they  yield  peptones  and  albumoses,  and  a 
phosphorised  residue  which  is  in  most  respects  identical  with 
nuclein,  but  does  not  appear  to  yield  products  of  the  xanthin 
series  when  decomposed  by  acids.  They  are,  like  the  globulins, 
precipitated  from  solution  by  neutral  salts,  —  the  precipitate  be- 
coming swollen  and  slimy  when  the  precipitant  is  sodium  chloride 
or  magnesium  sulphate,  but  not  so  when  sodium  sulphate  is 
employed. 

It  is  impossible  as  yet  to  give  any  general  method  of  separating 
the  nucleo-albumins  from  the  parent  protoplasm.  Eeference  to 
the  works  quoted  below  is  essential  when  dealing  with  any  inves- 
tigation as  to  their  presence  in  particular  cases. 

When  casein  is  digested  with  pepsin  a  residue  of  nuclein  is 
left ;  and  it  appears  probable  that  casein  may  be  in  reality  a  com- 
pound of  this  substance  with  a  proteid,  or  that  it  is  a  nucleo- 
albumin.i  Egg-yolk  is  also  considered  by  some  authors  to  contain 
nuclein  as  a  nucleo-albumin,  which  is  further  stated  to  be  ferru- 
ginous,^ but  by  others  the  yolk  is  spoken  of  as  yielding  only 
nuclein.  Whichever  view  be  correct,  the  nuclein  of  yolk  does 
not  yield  members  of  the  xanthin  series  by  decomposition  with 
acids,^  —  resembling  in  this  respect  the  nuclein  from  milk.  Syn- 
ovial fluid  *  and  bile  (?)  ^  are  also  stated  to  contain  substances 
which,  though  resembling  mucin  in  physical  properties,  are  prob- 
ably nucleo-albumins. 

It  may  be  pointed  out  that  in  some  of  the  above  cases  the 
nucleo-albumin  is  obtained  from  non-nuclear  sources.  When,  on 
the  other  hand,  aqueous  extracts  are  made  of  certain  nucleated 
structures,  there  is  evidence  that  apart  from  the  nuclein  of  the 
nuclei,  some  nucleo-albumin  is  obtained  whose  presence  is  referred 
rather  to  the  cell-protoplasm  than  to  the  nuclei :  this  is  the  case 
with  liver-cells,^  the  cells  of  the  submaxillary  gland,"  and  lymph- 
corpuscles.^  Non -nucleated  red  blood-corpuscles  do  not  yield  any 
nucleo-albumin.^ 

1  Lubavin,  Hoppe-Seyler's  Med.-chem.  Unters.  Hf.  iv.  (1871),  S.  463.  See  also 
Ber.  d.  deiitsch.  chem.  Gesell.  1877,  S.  2238.  Hamraarsten,  Zt.  f.  physiol.  chem.  Bd. 
VII.  (1883),  S.  273. 

2  Bunge,  Zt.  f.  -plui&wl.  Chem.  Bd.  ix.  (1885),  S.  49.  See  also  his  Text-book, 
p.  100. 

3  Kossel,  Arch.  f.  Physiol.  Jahrg.  1885,  S.  346. 

*  Hammarsten  (Swedish).     See  Abst.  in  Maly's  Ber.  Bd.  xii.  (1882),  S.  480. 

5  Paijiiull,  Zt.  f.  physiol.  Chem.  Bd.  xii.  (1888),  S.  196. 

6  Pldsz,  Pliiger's  Arch.  Bd.  vii.  (1873),  S.  371.  Hammarsten,  Ibid.  Bd.  xxxvi. 
(1885),  S.  351. 

■^  Hammarsten,  Zt.  f.  physiol.  Chem.  Bd.  xii.  (1888),  S.  174. 

8  Halliburton,  Jl.  of  Physiol.  Vol.  ix.  (1888),  p.  235. 

9  Halliburton  and  Friend,  Ibid.  Vol.  x.  (1889),  p.  543. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.  91 


CAKBOHYDEATES.i 

Certain  members  only  of  this  extensive  class  have  been  found 
in  the  human  body ;  of  these,  the  most  important  and  wide-spread 
are  glycogen,  grape-sugar  or  dextrose  (glucose),  with  which 
diabetic  sugar  seems  to  be  identical,''^  maltose,  and  milk-sugar. 
Inosit,  which  has  the  same  percentage  composition  as  a  sugar 
(CgHiaOg)  and  possesses  a  distinctly  sweet  taste,  has  hence  been 
usually  classed  with  the  carbohydrates.  This  is  incorrect,  since 
it  is  now  known  to  belong  to  the  benzol  series  (see  below,  p. 
108). 

Although  the  above-mentioned  carbohydrates  may  be  detected 
in  various  tissues  and  secretions  of  the  animal  body,  their  presence 
in  the  several  cases  is  not  so  much  due  to  their  introduction  into 
the  body  in  the  form  in  which  they  there  occur  as  to  their  pro- 
duction from  other  members  of  the  carbohydrate  group  existing 
in  food.  The  chief  of  these  is  starch,  and  it  will  perhaps  conduce 
to  completeness  to  deal  first  very  briefly  with  this  parent-sub- 
stance and  some  of  the  products  of  its  decomposition. 

The  Staech  Gkoup. 
1.     Starch  (C6Hio05)„. 

Starch  occurs  characteristically  in  plants  and  is  formed  in  their 
green  parts  under  the  determinant  influence  of  the  chlorophyll- 
corpuscles.  The  exact  mode  of  its  formation  is  however  as  yet 
undecided.  It  exists  in  plant-tissues  in  the  form  of  grains  which 
vary  in  size  and  shape  according  to  the  plant,  but  which  possess 
the  common  characteristic  of  exhibiting  a  stratified  structure, 
which  is  much  more  marked  in  some  cases  (potato-starch)  than 
in  others,  and  the  phenomena  of  double-refraction  when  examined 
in  polarised  light.  Considered  as  a  whole  the  grains  appear  to 
be  composed  of  two  substances  of  which  the  chief  both  in  quan- 
tity and  importance  is  called  granulose  and  the  other  cellulose. 
The  former,  which  yields  the  blue  colour  characteristic  of  starch 
on  the  addition  of  iodine,  may  be  dissolved  out  by  the  action  of 
saliva  or  malt-extract,  leaving  a  cellulosic  skeleton  of  the  original 
grain.  This  so-called  cellulose  is  not  identical  with  ordinary 
cellulose,  as  shown  by  its  ready  solubility  in  several  reagents 
which  do  not  dissolve  the  latter.^  When  treated  with  boiling 
water  the  grains  swell  up  and  finally  burst,  yielding  a  uniform 
viscous  mass  of  starch-paste  of  which  the  chief  component  is  the 

1  The  carbohydrates  are  very  fully  treated  in  Tollens'  Hdbch.  d.  Koldenhijdi-ate, 
Breslau,  1888.     See  also  Miller's  Cheinistri/,  Pt.  iii.  Sec.  1  (1880),  p.  567  et  seq. 

-  There  is  perhaps  some  slight  doubt  as  to  this  identity,  based  chiefly  upon  a 
slight  apparent  difference  in  the  specific  rotatory  power  of  true  dextrose  and  that 
obtained  from  diabetic  urine.     (See  Miller's  Chemistn/,  p.  583.) 

3  Brown  and  Heron,  Jl.  Ch.  Soc.  Vol.  xxxv.  (1879),  p.  611.  Liebig's  Aim.  Bd. 
cxcix.  S.  165. 


92  STAKCH. 

granulose.  The  mass  thus  obtained  cannot  be  regarded  as  a  true 
sclution  of  starch,  and  it  filters  with  extraordinary  difficulty, 
leaving  a  gelatinous  residue  on  the  filter,  however  dilute  the 
starch-paste  may  be  which  is  used  for  the  filtration.  When  sub- 
jected to  hydrolytic  agencies  such  as  superheated  water,  dilute 
acids  and  enzymes  the  starch  passes  rapidly  into  true  solution, 
yielding  at  the  same  time  a  series  of  successive  products  to  be 
described  below. 

Many  attempts  have  been  made  to  assign  a  definite  formula  to 
this  substance.  The  outcome  of  these  is  that  the  molecule  of 
starch  is  certainly  not  CeHioOs  but  n  (CeHioOs),  where  7i  is  not 
less  than  5  or  6  and  is  probably  much  larger. 

When  starch  is  converted  into  dextrose  by  treatment  with  dilute 
boiling  sulphuric  acid,  it  is  found  that  99  parts  of  starch  yield  108  of 
dextrose.^     Thus 

[(C6Hio05)6  +  H2O]  (mol.  =  990)  +  SH^O  ==  eCgHiaOe  (mol.  =  1080). 

Most  recently,  and  in  continuation  of  previous  researches,  it  has 
been  shown,  by  an  apjjlication  of  Eaoult's  method,  that  the  molecule 
of  soluble  starch  must  probably  be  represented  by  the  formula  5  (C12 
H2oOio)2o-^  Formulae  based  on  analyses  of  the  supposed  compound  of 
starch  with  iodine  are  probably  valueless,  since  there  is  but  little  rea- 
son to  suppose  that  any  such  definite  compound  exists, 

2.     Soluble  starch  (Amylodextrin)  (C6Hio05)„. 

When  starch-paste,  heated  to  40°  C.  on  a  water-bath,  is  digested 
with  a  small  amount  of  saliva  and  the  whole  stirred  so  as  to 
effect  a  thorough  mixture  of  the  two,  the  paste  rapidly  loses  its 
opalescent  appearance,  becoming  limpid  and  clear  like  water : 
the  moment  this  change  has  taken  place  the  digesting  mixture 
should  be  boiled  to  cut  short  the  further  action  of  the  ptyalin. 
The  fluid  thus  obtained  contains  the  first  product  of  the  hydrolysis 
of  starch  to  which  the  name  of  '  soluble  starch '  has  been  given. 
Its  solution  filters  readily,  and  the  filtrate  yields  with  iodine  the 
pure  blue  characteristic  of  the  original  unaltered  starch.  On  the 
addition  of  an  excess  of  alcohol  the  soluble-starch  is  precipitated, 
the  precipitate  after  drying  being  but  little  soluble  in  cold  water 
although  it  readily  dissolves  in  water  at  60 — 70°  C.  It  also 
yields  a  characteristic  precipitate  with  tannic  acid,  and  differs  in 
this  respect  from  the  dextrins.^     It  is  dextrorotatory 

(a)i,  =  +  194-8°  [(a)i  =  216°l 

and  does  not  reduce  Fehling's  fluid.     The  same  substance  may  be 

1  Sachsse,  Sitzb.  d.  Natforsch.  Gesell.  Leipzig,  1877.  Chem.  Centralb.  1877, 
No.  46. 

^  Brown  and  Morris,  J  I.  Chem.  Soc.  Vol.  lv.  July,  1889,  p.  462. 
3  Gries.smayer,  Annal.  d.  Chem.  Bd.  clx.  (1871),  S-  40. 


CHEMICAL   BASIS   OF  THE  ANIMAL   BODY.  93 

similarly  obtained  by  the  limited  action  of  malt-extract  or  pan- 
creatic juice. 

3.     Thedextrins  (CeHioOs)^.^ 

When  the  hydrolytic  action  of  saliva,  malt-extract,  or  pancreatic 
juice  on  starch-paste  is  prolonged,  the  first-formed  soluble-starch 
is  rapidly  changed  into  a  number  of  successive  substances  to  which 
the  general  name  of  dextrin  is  given.  These  products  are  inter- 
mediate between  soluble-starch  and  the  sugars  which  result  from 
the  complete  hydrolysis  of  starch,  and  are  probably  very  numer- 
ous, the  similarity  in  the  properties  of  the  successively  formed 
dextrins  rendering  their  separation  and  characterisation  extremely 
difficult.  They  are  all  precipitable  by  alcohol,  and  differ  from 
soluble-starch  in  yielding  no  precipitate  with  tannic  acid. 

(i)  Erythi-odextrin.  If  during  the  earlier  stages  of  the  hydro- 
lysis of  starch-paste,  successive  portions  of  the  solution  be  tested 
by  the  addition  of  iodine,  it  may  be  observed  that  the  pure  blue 
which  it  yields  at  first  passes  gradually  through  violet  and  red- 
dish-violet to  reddish-brown,  the  latter  colour  being  supposedly 
due  to  the  presence  in  the  solution  of  erythrodextrin,  whence  the 
name.  But  little  is  definitely  known  of  this  dextrin  as  a  chemical 
individual,  its  chief  characteristic  being  the  colour  it  yields  with 
iodine.^  The  violet  observed  during  the  earlier  stages  of  hydro- 
lysis is  due  to  an  admixture  of  the  blue  due  to  soluble-starch 
with  the  red  of  the  erythrodextrin. 

Commercial  dextrin,  which  is  very  impure,  containing  dextrose  and 
frequently  unaltered  starch,  usually  yields  a  very  strong  red  coloura- 
tion of  the  addition  of  iodine. 

(ii)  Achroodextrin.^  When,  during  the  prolonged  enzymic 
hydrolysis  of  starch  under  ordinary  conditions,  the  addition  of 
iodine  ceases  to  give  any  colouration,  the  fluid  now  contains  much 
sugar  together  with  a  considerable  but  variable  proportion  of  this 
dextrin,  which  has  received  its  name  from  its  behaviour  towards 
iodine,  yielding  no  colour  with  this  reagent.  It  is  the  character- 
istic dextrin  obtained  during  the  prolonged  artificial  digestion  of 
starch  with  saliva  (or  pancreatic  juice)  and  may  be  separated 
from  its  solution  by  concentration  and  the  addition  of  an  excess 
of  alcohol.  As  thus  prepared  it  is  mixed  with  much  adherent 
maltose  (see  below),  from  which  it  cannot  be  entirely  freed  by 
washing  with  alcohol  or  by  successive  solution  in  water  and 
reprecipitation  with  alcohol.     A  partial  separation  may  be  ob- 

^  For  the  probable  value  of  n  in  certain  cases,  see  Brown  and  Morris,  cit.  sub 
starch. 

2  But  see  Musculus  u.  Meyer,  Zt.  physiol.  Chem.  Bd.  iv.  (1880),  S.  451. 
"  Brown  and  Morris,  //.  Ch.  Soc.  Vol.  xlvii.  (1885),  p.  551. 


94  DEXTRIN. 

tained  by  fermenting  off  the  sugar  with  yeast  (O'Sullivan)  or  by 
dialysis,  since  dextrin  is  non-diffusible.  If  however  the  mixture 
be  warmed  with  a  slight  excess  of  mercuric  cyanide  and  caustic 
soda,  the  whole  of  the  sugar  is  destroyed  in  reducing  the  mercuric 
salt,  leaving  in  solution  a  non-reducing  dextrin. ^  As  thus  pre- 
pared it  appears  to  possess  a  constant  dextrorotatory  power  (a.)D  = 
194-8°  [(a)j=216°],  and  as  precipitated  by  alcohol  is  a  white 
amorphous  powder  very  soluble  in  water. 

Maltodextrin.'^  This  substance  is  described  as  appearing  during 
the  earlier  stages  of  a  limited  hydrolysis  of  starch-paste  with  diastase, 
and  it  may  perhaps  similarly  occur  when  saliva  or  pancreatic  juice 
is  employed.  It  differs  from  the  dextrins  previously  described  as 
follows.  It  is  more  soluble  in  alcohol  and  distinctly  diffusible;  it 
reduces  Peliling's  fluid,  has  a  lower  specific  rotatory  power 

(a)i,  =  + 174 -2°  [(«),=  193 -r], 

and  is  completely  convertible  into  maltose  by  the  further  action  of 
diastase.  It  will  therefore  not  be  found  among  the  products  of  a  pro- 
longed hydrolytic  degradation  of  starch. 

When  starch-paste  is  hydrolysed  outside  the  body  with  diastase 
or  with  animal  enzymes  some  dextrin  is  always  obtained  together 
with  the  sugars  which  make  their  characteristic  appearance  dur- 
ing the  process.  Considerable  difference  of  opinion  has  been 
expressed  as  to  the  possibility  of  a  complete  conversion  of  these 
dextrins  into  sugar  by  the  renewed  action  of  the  enzyme  upon 
them  after  their  isolation,  but  the  balance  of  opinion  appears  to 
be  that  the  conversion  is  in  many  cases  either  impossible  or  takes 
place  with  slowness  and  difficulty.  If  this  is  so  then  the  course 
of  an  artificial  and  normal  digestion  of  starch  is,  as  regards  the 
final  products,  very  different  in  the  two  cases,  for  there  is  no  evi- 
dence that  in  the  body  any  carbohydrate  is  absorbed  as  dextrin 
from  the  alimentary  canal.  The  conditions  however  under  which 
the  two  digestions  are  carried  on  are  markedly  different,  and 
more  particularly  with  respect  to  the  very  complete  and  continu- 
ous removal  of  digestive  products  in  the  natural  process  as  com- 
pared with  their  accumulation  in  an  ordinary  artificial  digestion. 
Now  there  is  no  doubt  that  the  products  of  an  enzymic  hydrolysis 
are  inhibitory  to  the  further  action  of  the  enzyme,^  and  this  is 
probably  the  cause  of  the  observed  difference.  In  accordance 
with  this,  if  a  starch  digestion  be  carried  on  in  an  efficient  dia- 
lyser,  the  starch  may  be  practically  entirely  converted  into  sugar, 
the  small  residue  of  dextrin  being  due  rather  to  inefficiency  of  the 

^  It  should  be  carefully  borne  in  mind  that  probably  many  forms  of  dextrin  exist, 
especially  among  the  earlier  products  of  hydrolysis,  none  of  which  give  any  colour- 
ation with  iodine. 

^  Brown  and  Morris,  loc.  c!t.  p.  561. 

3  See  also  Lindet,  Compt.  Rend.  T.  cviii.  (1889),  p.  453,  with  special  reference  to 
maltose. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.  95 

apparatus  than  to  the  chemical  resistance  of  the  dextrins  to  com- 
plete conversion  into  sugar.^  Although  this  statement  is  based 
upon  experiments  made  with  saliva,  there  is  no  reason  to  suppose 
that  the  same  will  not  hold  good  in  the  case  of  the  pancreatic 
juice  by  whose  action  the  chief  carbohydrate  digestion  of  the 
body  is  carried  on.  We  shall  therefore  not  be  far  wrong  in  con- 
cluding that  in  the  animal  body  starch  is  completely  converted 
into  sugar  previous  to  absorption,  and  if  this  be  the  case  the 
interest  of  the  physiologist  in  the  primary  products  of  starch 
hydrolysis  becomes  very  small,  except  so  far  as  a  study  of  these 
products  is  essential  to  the  elucidation  of  the  probable  molecular 
magnitude  and  structure  of  the  parent-substance. 

When  starch  is  treated  with  dilute  boiling  acids,  the  products 
which  have  been  so  far  described  are  formed  in  rapid  succession, 
the  whole  being  finally  converted  into  dextrose. ^ 

4.  Animal-gum  (C12H20O104-2H2O)  (?). 

This  is,  according  to  Landwehr,  a  form  of  carbohydrate  which 
may  be  extracted  by  the  prolonged  action  of  superheated  water 
from  salivary  and  mucous  glands,  and  is  found  also  in  milk  and 
urine.  It  has  already  been  briefly  described  above  (p.  79),  where 
its  chief  characteristics  have  been  given.  To  these  may  here  be 
added  that  it  yields  no  colouration  with  iodine,  is  very  feebly 
dextrorotatory  and  appears  to  form  a  compound  with  cupric 
oxide ;  the  latter  is  obtained  when  caustic  soda  and  sulphate  of 
copper  are  added  to  its  solution,  and  may  be  used  for  the  separa- 
tion of  animal-gum  from  urine.^ 

5.  Glycogen  (CeHioO-)^. 

This  substance  is  from  a  purely  chemical  point  of  view  ex- 
tremely like  starch,  the  similarity  being  most  marked  when  the 
hydrolytic  products  of  the  two  are  compared.  A  study  of  its  oc- 
currence, behaviour,  and  fate  in  the  animal  body  leaves  but  little 
doubt  that  it  may  be  regarded  from  the  physiological  side  as  truly 
the  animal  analogue  of  the  vegetable  starch,  and  as  such  it  is  fre- 
quently spoken  of  as  '  animal  starch.'  It  was  first  described  as  a 
constituent  of  the  liver  by  Bernard*  and,  simultaneously  though 
independently,  by  Hensen.^  In  more  recent  times  it  has  been 
found  to  occur  in  greater  or  less  quantities  in  many  tissues  of  the 

1  Lea,  Jl.  of  Physiol.  Vol.  xi.  (1890),  p.  226. 

2  But  see  Wohl,  Ber.  d.  d.  chern.  GeselL,  Jahrg.  xxiii.  (1890),  S.  2101. 

3  Landwehr,  Centralb.  f.  d.  Med.  Wiss.,  1885,  S.  369.  See  also  Wedenski,  Zt.f. 
physiol.  Chem.  Bd.  xiii.  (1889),  S.  122. 

*  Gaz.  med.  de  Pans,  1857,  No.  13.  Compt.  Rend.  T.  xliv.  (1857),  p.  579.  Gaz. 
Hebdom.  1857,  No.  28. 

s  Arch./,  path.  Anat.  u.  Physiol.  Bd.  xi.  (1857),  S.  395. 


06  GLYCOGEN. 

adult  body,  as  for  instance  the  muscles,^  also  in  white  blood-  and 
pus-corpuscles  ^  and  other  contractile  protoplasm  (Aethalium  sep- 
ticum),^  in  which  its  presence  is  significantly  connected  with  their 
specialised  activity,  not  as  an  essential,  as  some  have  supposed, 
but  as  a  convenient  accessory.  It  is  also  conspicuously  found  in 
the  tissues  of  the  embryo  before  the  liver  is  functionally  active,* 
and  is  present  in  large  quantities  in  many  moUusks,  as  for  in- 
stance the  common  oyster  ^  (9-5  p.c). 

It  is  at  present  uncertain  whether  the  glycogen  obtainable  from 
muscles  is  identical  with  that  of  the  liver.  It  is  stated  that  muscle- 
glycogen  yields  a  distinctly  more  purple  colour  with  iodine  than  does 
liver  gl^rcogen,^  but  their  identity  is  still  an  open  question.'' 

Preparation  of  glycogen.  The  liver  of  an  animal  (rabbit  or 
dog),  previously  fed  with  copious  meals  of  carbohydrate,  is  excised 
as  rapidly  as  possible,  cut  into  small  ^Dieces,  and  thrown  into  an 
excess  of  boiling  water,  at  least  400  c.c.  to  each  100  gr.  of  liver. 
After  being  boiled  for  a  short  time,  the  pieces  are  removed,  ground 
up  as  finely  as  possible  in  a  mortar  with  sand  or  powdered  glass, 
returned  to  the  original  water,  and  boiled  again  for  some  time.  On 
faintly  acidulating  the  boiling  mass  with  acetic  acid  a  large  amount 
of  the  proteid  matter  in  solution  is  coagulated  and  may  be  removed 
by  filtration.  The  filtrate  is  now  rapidly  cooled,  and  the  proteids 
finally  and  completely  precipitated  by  the  alternating  addition  of 
hydrochloric  acid  and  of  a  solution  of  the  double  iodide  of  mer- 
cury and  potassium  (Briicke's  reagent),^  as  long  as  any  precipi- 
tate is  formed.  The  precipitated  proteids  are  again  removed  by 
filtration,  the  glycogen  precipitated  by  the  addition  of  two  vol- 
umes of  95  p.c.  alcohol,^  collected  on  a  filter,  washed  thoroughly 
with  60  p.c.  spirit,  and  finally  with  absolute  alcohol  and  ether 
(Brlicke).io 

The  above  method  suffices  in  cases  where  there  is  mucli  glycogen 
present  and  no  quantitative  result  is  desired  ;  as  a  matter  of  fact 
there  is  a  not  inconsiderable  loss  during  its  application.  The  ac- 
curate determination  of  glycogen  in  tissues  is  a  matter  of  some 
difficulty,  primarily  because  it  is  not  easy  to  ensure  the  complete 
separation   into   solution  of  the   glycogen  from  the   tissue,   and  sec- 

1  Nasse,  Pfluger's  Arch.  Bd.  ii.  (1869),  S.  97. 

2  Hoppe-Sevler,  Med.-chem.  Unters.  Hft.  4  (1871),  S.  486. 

3  See  refs.  on  p.  4.     Also  Kiihne,  Physiol.  Chem.  1868,  S.  334. 

*  See  Preyer's  Specialle  Physiol,  d.  Embryo,  Leipzig,  1885,  S.  271. 

5  Bizio,  Compt.  Rend.  T.  lx'ii.  (1866),  p.  675. 

6  Naunyn,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  in.  (1875),  S.  97.  Boehm  u. 
Hoffmann,  Ibid.  Bd.  x.  (1878),  S.  12.     Nasse,  Pfluger's  Arch.  Bd.  xiv.  (1877),  S.  479. 

■^  See  also  Muscnlus  u.  v.  Mering,  Zt.f.  phj/siol.  Chem.  Bd.  ii.  (1878),  S.  417. 

^  Prepared  by  saturating  a  boiling  10  p.c.  soluti  of  potassium  iodide  with 
freshly  precipitated  iodide  of  mercury ;  on  cooling,  th.  is  filtered  and  the  filtrate 
employed  as  directed. 

^  So  that  the  mixture  contains  60  p.c.  of  alcohol. 

M  Sitsb.  d.  Wien.  Akad.  Bd.  lxiii.  (1871),  2  Abth.  Feb.-Hft.,  S.  214. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.         97 

ondarily  owing  to  a  possible  loss  during  the  precipitation  and  re- 
moval of  the  proteids  with  which  it  is  always  largely  contaminated. 
The  first  difficulty  may  be  largely  overcome  by  the  addition  of  caustic 
potash  which  dissolves  the  tissue  fragments  and  thus  liberates  the 
glycogen;  also  by  extraction  in  a  Papin's  digester/  in  which  case  tlie 
solution  is  again  very  complete.^ 

Glycogen  is,  when  pure,  an  amorphus  white  powder,  readily 
soluble  in  water  with  which  it  yields  a  solution  which  is  usu- 
ally, but  not  always,  opalescent.  This  solution  contains  no 
particles  which  are  visible  under  the  microscope  and  filters 
readily  without  diminution  of  the  opalescence ;  the  latter  may 
be  largely  removed  by  the  addition  of  free  alkalis  or  acetic  acid. 
Under  ordinary  conditions  it  is  readily  precipitated  by  alcohol 
when  the  mixture  contains  60  p.c.  of  the  precipitant,  but  if 
pure,  and  in  0-5 — I'O  p.c.  solution,  even  an  excess  of  absolute 
alcohol  is  stated  not  to  cause  its  precipitation.  The  precipita- 
tion takes  place  at  once  on  the  addition  of  a  trace  of  sodium 
chloride.^ 

It  gives  a  characteristic  port-wine  colouration  with  iodine,  which 
does  not  however  distinguish  it  from  erythrodextrin  since  in  both 
cases  the  colour,  contrary  to  the  older  and  current  statements,  dis- 
appears on  warming  and  returns  on  cooling.  On  the  other  hand, 
dextrins  are  not  precipitated  by  60  p.c.  alcohol,  even  the  most 
insoluble  of  these  substances  requiring  at  least  85  p.c.  of  alco- 
hol for  their  precipitation,  and  usually  more.  It  appears  that 
the  reaction  with  iodine  is  most  delicate  in  presence  of  sodium 
chloride.* 

Aqueous  solutions  of  glycogen  are  strongly  dextrorotatory,  but 
the  statements  as  to  its  specific  rotatory  power  must  be  received 
with  caution.  [^BoeJim  a7id  Hoffmann^  (a) j)  =  -\-226-7°.  Kiilz^ 
in  -6  p.c.  solution  {a~)j,  =  4-203-5°  to  +  225-6°.  Lmidwehr  7  (^a\  = 
+213-3°]. 

The  molecular  magnitude  of  glycogen,  like  that  of  starch,  is  un- 
known. Glycogen  yields  precipitates  with  tannic  acid,  also  with 
calcium  and  barium  hydrate, ^  and  with  basic  lead  acetate.  No  reli- 
ance can  however  be  placed  on  the  determination  of  the  molecular 
weight  of  glycogen  from  an  analysis  of  these  compounds. 

1  Boehm,  Pfliiger's  Arch.  Bd.  xxiii.  (1880),  S.  44. 

2  The  whole  subject  is  very  fully  treated  by  Kiilz  in  Zt.f.  Biol.  Bd.  xxii.  (1886), 
S.  161,  where  also  the  literature  is  comprehensively  quoted.  See  additionally  Nasse, 
Pfliiger's  Arch.  Bd.  xxxvii.  (1885),  S.  582,  and  Landwehr,  Ibid,  xxxviii.  S.  321. 
Panormow  (Polish).  See  Abst.  Maly's  Jahresh.  1887,  S.  304.  Cramer,  Zt.f.  Biol. 
Bd.  XXIV.  (1888),  S.  67. 

3  Kiilz,  Bcr.  d.  d.  chem.  Gesell.  Jahrg.  1882,  S.  1300. 

4  Nasse,  Pfliiger's  Arch.  Bd.  xxxvii.  (1885),  S.  585. 

5  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  vii.  (1877),  S.  489. 

6  Pfliiger's  Arch.  Bd.  xxiv.  (1881),  S.  85. 

7  Zt.  f.  phy.'^ioi.  Chem.  Bd.  viii.  (1883),  S.  170. 

»  Nasse,  Pfliiger's  Arch.  Bd.  xxxvii.  (1885),  S.  582. 

7 


98  GLYCOGEN. 

The  liyclrolytic  products  obtained  by  the  action  of  enzymes  and 
dilute  boiling  acids  on  glycogen  have  not  been  as  fully  studied  as 
they  have  in  the  case  of  starch,  but  the  general  course  of  the  de- 
composition is  the  same  in  both  cases.  Thus  when  treated  with 
dilute  mineral  acids  at  100°C.,  the  opalescence  disappears,  some 
dextrin  is  formed  en  passant,  and  finally  the  solution  contains 
only  dextrose.^  On  the  addition  of  saliva  or  pancreatic  juice  to 
a  solution  of  glycogen  at  40°,  the  first  change  observed  is  an  im- 
mediate disappearance  of  the  opalescence,  followed  by  a  rapid  con- 
version into  some  form  of  dextrin  and  a  considerable  proportion 
of  a  sugar  which  is  apparently  identical  with  maltose.^  Some 
trace  of  dextrose  may  perhaps  at  the  same  time  be  formed. 

The  change  which  glycogen  in  the  liver  undergoes  post-mortem 
and  presumably  also  during  life  is  strikingly  different  from  that 
which  has  been  described  above.  Whereas  by  ordinary  enzymic 
hydrolysis,  maltose  is  the  chief  final  product  obtained,  there  is 
now  no  doubt  that  in  the  liver  little  if  any  maltose  is  formed,  the 
so-called  liver-sugar  being  apparently  identical  with  true  dex- 
trose. This  fact  throws  considerable  light  on  the  mode  of  con- 
version of  glycogen  into  sugar  by  the  liver.  It  has  been  most 
usually  taught  that  this  conversion  is  due  to  some  fermentative 
action ;  if  this  were  so  then  the  enzyme  which  is  the  active  agent 
must  be  possessed  of  powers  differing  from  those  of  most  other 
enzymes  since  it  forms  dextrose  and  not  maltose.  But  as  a  mat- 
ter of  fact  it  does  not  appear  possible  to  extract  any  appreciable 
quantity  of  enzyme  from  the  liver,  and  if  a  trace  is  obtained  it  is 
of  one  whose  action  on  starch  and  glycogen  yields  chiefly  maltose 
and  not  dextrose.  It  is  hence  a  legitimate  conclusion  that  the 
conversion  of  glycogen  into  sugar  by  the  liver  is  the  outcome  of 
the  specific  metabolic  activity  of  the  hepatic  cells,  and  not  of  any 
enzymic  action.^  It  is  also  significantly  probable,  from  what  has 
been  already  said  (see  above,  p.  59),  that  the  liver  receives  its 
carbohydrates  supplied  in  the  form  of  dextrose,  and  there  is  no 
doubt  that  diabetic  sugar  is  closely  related  to,  if  not  identical 
with,  true  dextrose. 

The  dextrin  which  some  observers  have  obtained  from  muscles  is 
not  to  be  regarded  as  a  specific  constituent,  but  as  formed  from  their 
glycogen  by  some  post-mortem  change.  Horse-flesh  is  peculiarly  rich 
in  glycogen,  and  it  was  chiefly  from  this  source  that  dextrin  was  ob- 
tained in  large  amount.^ 

1  Maydl,  Zt.  f.  phijsiol.  Chem.  Bd.  in.  (1879),  S.  194.  Kulz  u.  Borntrager, 
Pfluger's  Arch.  Bd.  xxiv.  (1881),  S.  28.     Seegen,  Ibid.  Bd.  xix.  (1879),  S.  106. 

2  Musculus  u.  V.  Mering,  Zt.  f.  physiol.  Chem.  Bd.  ii.  (1878),  S.  403.  Seegen, 
loc.  cit.     Kulz,  Pfluger's  Arch.  Bd!  xxiv.  (1881),  S.  81. 

3  Eves,  Jl.  of  Physiol.  Vol.  v.  (1884),  p.  342  (contains  lit.  to  date).  See  more 
recently  Langendorff,  Arch.  f.  Physiol.  1886.  Suppl.-Bd.  S.  277.  Panormow,  Klin. 
Wochenb.  1887,  No.  27.     Dastre,  Arch,  de  Physiol.  (4)  T.  i.  (1888),  p.  69. 

*  Limpricht,  Liebig's  Ann.  Bd.  cxxxiii.  (1865),  S.  293. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.  99 


6.     Cellulose  (C^H.^O,).. 

Although  true  cellulose  is  never  found  as  a  constituent  of  the 
animal  tissues,  it  possesses  no  inconsiderable  interest  for  the  phys- 
iologist in  view  of  the  fact  that  in  the  herbivora  a  large  amount 
of  cellulose  is  digested  and  absorbed  so  that  it  does  not  reappear 
externally  in  the  excreta.  In  man  also  there  is  no  doubt  that 
some  digestion  and  absorption  of  cellulose  may  occur,  the  process 
being  facilitated  by  the  fact  that  in  those  more  succulent  vegeta- 
bles and  fruits  in  which  it  is  taken  by  man,  the  cell-walls  are 
comparatively  non-lignified  and  hence  more  easily  acted  upon  by 
the  digestive  agents. 

The  lignification  of  the  cell-wall  which  has  taken  place  in  those 
plant  tissues  to  which  the  name  '  woody '  is  ordinarily  applied  is  due 
to  the  presence  of  a  substance  called  lignin.  Very  little  is  known  of 
it  as  a  chemical  individual :  it  appears  to  contain  more  carbon  than 
does  cellulose.  Its  discrimination  from  cellulose  depends  on  the  fact 
that  it  is  coloured  yellow  by  the  action  of  Schulze's  reagent  (see  below) 
and  deep  brown  by  that  of  iodine  and  sulphuric  acid.  When  treated 
with  phloroglucin  and  strong  hydrochloric  acid  it  turns  red;  it  is 
coloured  bright  yellow  by  the  action  of  aniline  sulphate  or  chloride 
and  the  subsequent  addition  of  hydrochloric  acid. 

Further,  although  at  present  but  little  is  known  as  to  how  the 
digestion  of  cellulose  is  brought  about  in  the  alimentary  canal, 
there  is  increasing  evidence  of  the  possible  existence  of  a  specific 
enzyme  to  whose  solvent  action  the  change  is  due.  But  as  yet 
this  evidence  rests  almost  entirely  upon  experiments  with  and 
observations  of  vegetable  organisms.^ 

Cellulose  is  related  to  starch  and  in  some  cases  (Date,  Phytele- 
phas)  plays  the  part  of  a  store  of  reserve  material,  being  dissolved, 
presumably  by  some  enzyme,  and  utilised  during  germination. 
The  cell-wall  of  vegetable  cells  is  composed  of  cellulose,  which  in 
young  cells  is  pure  and  much  less  resistant  to  various  reagents 
than  it  is  in  the  older  cells  where  it  has  become  lignified  and 
incrusted  with  other  substances.  When  pure  it  is  soluble  in  one 
reagent  only,  viz.  Schweizer's  which  is  a  solution  of  hydrated 
cupric  oxide  in  ammonia.^  When  treated  with  strong  sulphuric 
acid  cellulose  is  changed  and  yields  a  substance  which  is  coloured 
blue  by  iodine ;  a  similar  colouration  is  observed  on  the  addition 

1  Brown  and  Morris,  Jl.  Chem.  Soc.  Vol.  lvii.  (1890),  p.  497.  Contains  refer- 
ences to  other  literature. 

-  Prepared  as  follows.  Sulphate  of  copper  in  solution,  to  which  some  ammonium 
chloride  has  been  added,  is  precipitated  with  caustic  soda :  the  hydrated  cupric  oxide 
thus  obtained  is  washed,  and  dissolved  to  saturation  in  20  p.c.  ammonia.  It  may 
also  be  prepared  by  pouring  strong  ammonia  on  to  copper  turnings,  the  requisite 
oxidation  of  the  copper  being  effected  by  drawing  a  current  of  air  through  the  fluid 
in  which  the  turnings  are  immersed.     (Cross  and  Bevan,  Cellulose,  1885,  p.  6.) 


100  CELLULOSE. 

of  iodine  after  the  action  of  chloride  of  zinc  (Schulze's  reagent). ^ 
These  reactions  afford  a  means  of  detecting  cellulose. 

By  treatment  with  strong  sulphuric  acid  cellulose  may  be  dis- 
solved with  the  formation  of  a  dextrin-like  product :  on  diluting 
with  water  and  boiling  it  is  finally  converted  into  a  sugar  which 
is  apparently  identical  with  dextrose.^ 

As  already  stated  cellulose  is  undoubtedly  digested  in  the  ali- 
mentary canal  more  especially  of  herbivora,  but  also  to  a  less  ex- 
tent of  man.^  We  know  however  but  little  of  the  real  nature  of 
the  digestive  processes  which  are  involved  in  this.  Two  views 
are  open  to  us.  It  has  long  been  known  that  under  the  influence 
of  putrefactive  organisms,  as  from  sewer-slime,  cellulose  is  disin- 
tegrated and  dissolved  with  an  evolution  of  marsh-gas  and  car- 
bonic anhydride.*  This  is  usually  known  as  the  marsh-gas  fer- 
mentation of  cellulose.  In  accordance  with  this  it  is  possible  that 
a  similar  factor  is  at  work  in  the  alimentary  canal,  more  especially 
of  the  herbivora  with  their  large  caecum  in  which  the  food  stays 
for  some  time.  This  accords  with  the  marked  occurrence  of  marsh- 
gas  in  the  gases  of  their  intestine  and  its  increased  presence  in  the 
intestine  of  man  when  largely  fed  with  a  vegetable  diet.^  On  the 
other  hand  it  is  possible  that  the  digestion  may  turn  out  to  be  due 
to  some  definite  enzyme,^  but  as  yet  no  such  enzyme  has  been 
obtained  with  certainty  from  the  secretions  or  tissues  of  the 
alimentary  canal.  Possibly  the  organisms  which  as  stated  above 
can  cause  the  decomposition  of  cellulose  do  so  by  means  of  some 
specific  enzyme.  It  remains  for  further  research  to  throw  a 
decisive  light  on  the  possibilities  to  which  attention  has  been 
drawn. 

Some  difference  of  opinion  exists  as  to  the  physiological  sig- 
nificance of  cellulose  digestion.  There  is  at  present  no  evidence 
that  the  cellulose  of  food  as  such  is  a  food-stuff  in  the  same  sense 
that  starch  is.  As  far  as  the  existing  evidence  goes  we  shall  not 
perhaps  be  far  wrong  in  supposing  that  cellulose  digestion  is 
primarily  important  as  liberating  from  the  cells  the  true  food- 
stuffs which  they  contain.     At  the  same  time  the  products  formed 

1  The  reagent  used  is  prepared  as  follows.  Iodine  is  dissolved  to  saturation  in  a 
solution  of  chloride  of  zinc,  sp.gr.  rs,  to  which  6  parts  of  potassium  iodide  have  been 
added.  See  also  Bower,  Pi-act.  Bot.,  1891,  p.  506.  Cross  and  Bevan  (loc.  cit.  p.  7) 
recommend  the  following.  Zinc  is  dissolved  to  saturation  in  hydrochloric  acid,  and 
the  solution  evaporated  to  sp.gr.  2-0 ;  to  90  parts  of  this  solution  are  added  6  parts 
of  potassium  iodide  dissolved  in  10  parts  of  water,  and  in  this  solution  iodine  is  finally 
dissolved  to  saturation. 

2  Flechsig,  Zt.f.  Physiol.  Chem.  Bd.  vii.  (1883),  S.  523. 

3  Bunge,  Physiol,  and  Path.  Chem.  1890,  pp.  81,  191. 

4  Popoff,  Pfiiiger's  Arch.  Bd.  x.  (1875),  S.  113.  Van  Tieghem,  Compt.  Rend.  T. 
Lxxxviii.  (1879),  p.  205.  Hoppe-Seyler,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  xvi.  (1883), 
S.  122.     Zt.f.  physiol.  Ch.  Bd.  x.  (1886),  Sn.  201,  401. 

s  Tappeiner,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  xv.  (1882),  S.  999 ;  xvi.  Sn.  1734,  1740. 
Zt.  f.  Biol.  Bd.  XX.  (1884),  S.  52.  (Gives  literature  to  date.)  Jbid.  S.  215;  xxiv. 
(1888),  S.  105. 

6  Hofmeister,  Arch.  f.  Thierheilk.  Bd,  vii.  (1881),  S.  169;  xi.  (1885),  Hfte.  1,  2. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        101 

during  the  solution  of  the  cellulose  may,  if  they  are  oxidised  in 
the  body,  contribute  to  its  energy  and  thus  be  of  use.^ 

7.    Tunicin  (C6Hio05)„. 

This  substance  constitutes  the  chief  part  of  the  mantle  of  Tuni- 
cata  (Ascidians)  and  appears  to  have  been  first  described  by  C. 
Schmidt,^  who  drew  attention  to  its  similarity  to  vegetable  cellu- 
lose. This  view  was  confirmed  by  Berthelot,  who  however  observed 
that  it  is  much  more  resistant  to  the  action  of  acids  than  is  true 
cellulose.^  In  other  respects  the  two  may  be  regarded  as  identical. 
In  accordance  with  this  it  is  found  that  tunicin  is  soluble  in 
Schweizer's  reagent  (see  above),  from  which  it  may  be  reprecip- 
itated  by  hydrochloric  acid  and  thus  purified.  It  is  further 
coloured  blue  by  the  addition  of  iodine  after  preliminary  treat- 
ment with  sulphuric  acid.  It  is  soluble  in  concentrated  sulphuric 
acid,  and  if  water  be  added  to  this  solution  and  it  be  boiled  for 
some  time,  a  sugar  which  is  apparently  identical  with  ordinary 
dextrose  is  obtained.* 

It  is  prepared  in  the  pure  form  by  treating  the  mantles  for  some 
days  with  water  in  a  Papin's  digester,  then  in  succession  with 
boiling  dilute  hydrochloric  acid,  strong  caustic  potash  and  water. 
As  thus  obtained  it  retains  the  form  of  the  parent  tissue. 

The  Sugaks. 

The  researches  of  Emil  Fischer  have  thrown  a  fiood  of  light  on 
the  chemistry  of  the  sugars.^     In  phenyl-hydrazin  (CeHs.NH.NHa) 

he  discovered  a  reagent  which  forms  with  the  sugars  compounds 
known  as  hydrazones  and  osazones.  These  provided  for  the  first 
time  by  their  various  solubilities,  melting-points,  and  rotatory 
powers  an  adequate  means  of  detecting,  separating,  and  character- 
ising the  several  members  of  this  class  of  carbohydrates.  Hence 
it  became  possible  to  investigate  the  occurrence  of  sugars  among 
the  complicated  products  of  the  reactions  employed  in  the  effort 
to  effect  their  transformations  and  synthetic  production.  It  would 
be  out  of  place  here  to  enter  into  the  details  of  Fischer's  work,  and 
it  must  suffice  to  say  that  he  has  not  merely  synthetised  both 

1  On  the  above  see  Weiske,  Chem.  Centralb.  Bd.  xv.  (1884),  S.  385.  Henneberg 
u.  Stohmann,  Zt.f.  Biol.  Bd.  xxi.  (1885),  S.  613.  Weiske  (Ref.)  Schulze  u.  Flechsig, 
Ibid.  XXII.  S.  373. 

2  Liebig's  Ann.  Bd.  liv.  (1845),  S.  318. 

3  Ann.  d.  Ch.  et  Phjs.  3  Se'r.  T.  lvi.  (1859),  p.  149. 

4  Franchimont,  Ber.  d.  d.  chem.  Gesell.  1879,  S.  1938.  CompU  Rend.  T.  Lxxxix. 
(1879),  p.  75.5.     Schafer,  Liebig's  Ann.  Bd.  clx.  (1871),  S.  312. 

5  Fischer  has  given  a  condensed  account  of  his  researches,  with  full  references  to 
the  literature,  in  Ber.  d.  d.  chem.  Gesell.  Jahrg.  xxiii.  (1890),  S.  2114.  Of  this  an 
abstract  is  given  in  .//.  Chem.  Soc.  Nov.  1890,  p.  1223.  See  also  Schulz,  Biol.  Centralb. 
Bd.  x.  (1890),  Sn.  551,  620. 


102  DEXTROSE. 

dextrose  and  laBvulose,  and  definitely  established  the  fact  that  they 
are  respectively  an  aldehyde  and  ketone  of  the  hexacid  alcohol 
C6H8(OH)6,  but  has  in  addition  succeeded  in  producing  artificial 
sugars  containing  seven,  eight,  and  nine  carbon  atoms/  In  connec- 
tion with  the  latter  an  interesting  question  arises  as  to  the  prob- 
able effects  on  animal  metabolism  of  their  introduction  into  the 
body  instead  of  the  natural  sugars. 

The  osazones.  The  compounds  of  the  sugars  to  which  this 
generic  name  is  applied  are  formed  when  solutions  of  the  sugars 
are  warmed  for  some  time  on  a  water-bath  with  phenyl-hydrazin 
and  dilute  acetic  acid,  and  separate  out  either  in  an  amorphous  or 
crystalline  state.  Their  formation  takes  place  in  two  stages.  In 
the  first  the  sugar  combines,  as  do  the  aldehydes  and  ketones,  with 
one  molecule  of  the  base  to  form  a  compound  which  is  in  most  cases 
readily  soluble  and  is  known  as  a  hydrazone.  In  the  second  stage 
the  first-formed  hydrazone  is  oxidised  by  the  excess  of  phenyl- 
hydrazin  present,  and  the  substance  thus  produced  unites  with 
another  molecule  of  the  base  to  form  the  osazone.  As  already 
stated  the  osazones  of  the  various  sugars  differ  characteristically 
in  their  solubilities,  melting-points,  and  rotatory  powers.  They 
hence  afford  an  invaluable  means  not  only  for  detecting  and  iso- 
lating the  sugars,  but  also  for  discriminating  between  sugars  whose 
optical  and  reducing  powers  may.  afford  an  insufficient  distinction. 
Further,  in  some  cases  the  osazones  have  provided  a  means  of 
ascertaining  the  molecular  formula  of  certain  sugars  and  of  deter- 
mining the  constitution  of  others.  The  characteristic  properties 
of  the  several  osazones  are  given  below  under  the  respective  sugars. 


The  Dexteose  GtEoup. 

1,     Dextrose  (Glucose,  Grape-sugar). 

CsHiA-     [COH-(CH.OH)4-CH2.0H]. 

Is  found  in  minute  but  fairly  constant  quantities  as  a  normal 
constituent  of  blood,  lymph,  and  chyle.  Its  occurrence  in  the 
liver  has  been  already  referred  to  (§  465)  in  connection  with 
diabetes,  a  disease  which  is  characterised  by  the  large  amount  of 
dextrose  which  is  present  in  the  blood  and  the  still  larger  amount 
in  the  urine.  The  question  whether  dextrose  is  a  normal  constit- 
uent of  urine  has  led  to  much  dispute,  but  it  now  appears 
probable  that  it  is  present  in  minute  amounts.^  The  experimental 
difficulties  of  detecting  traces  of  sugar  in  this  excretion  are  con- 
siderable.    There  is  no  dextrose  normally  in  bile. 

1  Fischer  u.  Passmore,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  xxiii.  (1890),  S.  2226. 

2  For  literature  and  results  see  Neubauer  u.  Vogel,  Analyse  des  Hams  (Ed.  ix. 
1890),  S.  41. 


CHEMICAL   BASIS   OF  THE  ANIMAL  BODY.         103 

The  probability  that  it  is  as  dextrose  that  the  carbohydrates  are 
finally  absorbed  from  the  alimentary  canal  has  already  been  re- 
ferred to  (p.  59).  This  corresponds  with  the  fact  that  dextrose 
is  the  most  readily  assimilable  sugar,  as  is  known  from  compara- 
tive injections  of  the  various  sugars  into  the  blood-vessels  and 
observations  on  their  subsequent  appearance  in  the  urine. 

When  pure,  dextrose  is  colourless  and  crystallises  from  its 
aqueous  solution  in  six-sided  tables  or  prisms,  often  agglomerated 
into  warty  lumps.  The  crystals  will  dissolve  in  their  own  weight 
of  cold  water,  requiring  however  some  time  for  the  process ;  they 
are  very  readily  soluble  in  hot  water.  Dextrose  is  somewhat 
sparingly  soluble  in  cold  ethyl-alcohol,  more  soluble  in  warm; 
slowly  soluble,  but  in  considerable  quantity,  in  methyl-alcohol, 
and  insoluble  in  ether. 

It  may  be  prepared  by  concentrating-  diabetic  urine  until  it 
yields  crystals  of  dextrose ;  these  are  then  purified  by  recrystal- 
lisation  from  methyl-alcohol.  It  may  also  be  conveniently  pre- 
pared by  the  action  of  hydrochloric  acid  on  cane-sugar  dissolved 
in  alcohol.!  A  freshly  prepared  cold  aqueous  solution  of  dextrose 
possesses  a  specific  rotatory  power  for  monochromatic  yellow 
light  of  (a)n  =-\-  100°.  This  rapidly  falls,  especially  on  warm- 
ing, until  it  may  be  taken  as  (a)^  =+  52-5°  for  solutions  which 
do  not  contain  more  than  10  p.c.  of  the  sugar.  The  rotation  is 
however  dependent  on  the  concentration  of  the  solution  being 
least  with  very  dilute  solutions. 

The  specific  rotatory  power  of  a  substance  is  the  amount,  measured 
in  degrees,  by  which  the  plane  of  polarised  light  is  rotated  by  a  solu- 
tion which  contains  1  gram  of  the  substance  in  each  1  c.c.  when  ex- 
amined in  a  layer  1  dcm.  in  thickness.  Since  the  amount  of  rotation 
produced  in  any  given  case  is  directly  proportional  to  the  specific 
rotatory  power,  also  to  the  weight  of  substance  in  solution  and  the 
thickness    of    the    fluid    layer    in   which    it    is    examined   we    have 

a  =  (a)  X  i'  X  ^   or    (a)  = ,   where    (a)    is    the   specific  rotatory 

p  .  I 
power,  p  is  the  weight  in  grams  of  the  substance  in  1  c.c.  of  the  solu- 
tion, I  is  the  thickness  in  decimetres  of  the  fluid  layer  and  a  is  the 
observed  rotation.  This  equation  provides  a  means  of  estimating 
sugars  quantitatively  by  measuring  the  rotation  produced  by  a  solu- 
tion of  unknown  concentration  in  a  layer  of  known  thickness,  the 
specific  rotatory  power  being  known. ^ 

The  instruments  employed  for  measuring  the  amount  of  rotation 
produced  by  an  optically  active  substance  are  known  generically  as 
Polarimeters.  In  one  class  of  these  instruments  the  source  of  light 
used  is  a  brightly  luminous  sodium-flame,   the   determination   being 

1  Soxhlet,  Jn.f.prakt.  Chem.  (N.F.)  Bd.  xxi.  (1880),  S.  227. 

^  For  details  of  the  instruments  and  methods  see  Landolt,  Das  opt.ische  Drehungs- 
vermogen  organ.  Suhstanzen.  Hoppe-Seyler,  Physiol,  path.  chem.  Anal.  1883,  S.  24, 
Miller's  Chem.  (Ed.  by  Armstrong  and  Groves),  Pt.  iii.  1880,  p.  569  et  seq. 


104  DEXTROSE. 

made  for  the  monochromatic  light  corresponding  to  the  D  line  of  the 
solar  spectrum.  In  this  case  the  specific  rotatory  power  is  represented 
by  (a)^.  In  another  class  the  mean  yellow  light  of  an  argand  or 
paraffin  lamp  is  employed.  In  this  form  of  polarimeter  the  field  of 
the  instrument  when  adjusted  for  use  is  of  a  pale  pinkish-violet 
colour,  called  from  the  extreme  sensitiveness  with  which  it  changes 
from  pink  to  violet  or  the  reverse  the  'transition  tint'  (teinte  de 
passage) .  This  colour  is  complementary  to  yellow  (jaune),  and  specific 
rotatory  powers  determined  for  this  particular  colour  are  represented 
by  (a)j.     For  any  given  substance  (a)D  is  always  less  than  (a)j,  and 

for  ordinary  purposes  (a)^  =  -tTaq'  ^^'  ('^)d  •  (")j  •  •  21*65  :  24.     Hence 

it  is  important  in  all  cases  to  state  clearly  whether  a  given  determi- 
nation has  been  made  for  monochromatic  yellow  light  or  for  the  '  tran- 
sition tint '  of  mean  yellow  light. 

Dextrose,  like  all  alcohols,  readily  forms  compounds  with  acids 
and  many  salts  ;  of  these  the  latter  are  the  more  important  and  are  in 
many  cases  characteristic,  as  for  instance  those  with  caustic  alkalis 
and  sodium  chloride.  When  heated  many  of  these  compounds, 
more  particularly  those  of  bismuth,  copper,  and  mercury,  are  de- 
composed, the  decomposition  being  accompanied  by  the  precipita- 
tion either  of  the  metal  (Hg)  or  of  an  oxide  (CusO).  This  fact 
provides  the  basis  for  the  more  important  methods  of  detecting 
the  presence  of  dextrose  and  other  sugars  with  similar  reducing 
powers,  and  of  estimating  them  quantitatively  in  solution,  since 
it  is  found  that  the  amount  of  reduction  effected  by  any  given 
sugar  is,  under  given  conditions,  a  constant  quantity.^ 

Phenyl-glucosazone.     C18H02N4O4.     [C6H10O4  (CeH,.  N^H)^]. 

This  compound  of  dextrose  with  phenyl-hydrazin  crystallises  in 
yellow  needles.  It  is  almost  insoluble  in  water,  very  slightly 
soluble  in  hot  alcohol,  melts  at  about  205°,  and  is  Itevo-rotatory 
when  dissolved  in  glacial  acetic  acid.  The  phenyl-hydrazin  test 
for  dextrose  is  applied  as  follows.  To  50  c.c.  of  the  suspected 
fluid  (e.g.  diabetic  urine)  add  I — 2  grm.  hydrochloride  of  phenyl- 
hydrazin,  2  grm.  sodium  acetate,  and  heat  on  a  water-bath  for 
half  an  hour ;  or  else  add  10 — 20  drops  of  pure  phenyl-hydrazin 
and  an  equal  number  of  drops  of  50  p.c.  acetic  acid  and  warm  as 
before.^  On  cooling,  if  not  before,  the  glucosazone  separates  out 
as  a  crystalline  or  it  may  be  amorphous  precipitate.  If  amorphous 
it  is  dissolved  in  hot  alcohol,  the  solution  is  then  diluted  with 
water  and  boiled  to  expel  the  alcohol,  whereupon  the  compound 
is  obtained  in  the  characteristic  form  of  yellow  needles.     By  the 

1  The  description  of  the  various  methods  employed  for  the  detection  and  estima- 
tion of  dextrose  and  other  sugars  lies  outside  the  scope  of  this  work.  Full  details 
are  given  in  Neubauer  u.  Vogel,  Analyse  des  Harris,  and  ToUens'  Handbuch  der 
Kohlenh  i/drate. 

'^  Fischer,  Ber.  d.  d.  chem.  Gesell  Bd.  xxii.  (1889),  S,  90  (foot-note). 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.        105 

above  method  it  is  possible  to  obtain  tlie  crystals  from  fluids 
which  contain  only  0*5  grm.  per  litre. 

An  important  property  of  dextrose  is  its  power  of  undergoing 
fermentations.  Of  these  the  principal  are  :  (1)  Alcoholic.  This  is 
produced  in  aqueous  solutions  of  dextrose,  under  the  influence  of 
yeast.  The  decomposition  is  the  following :  CeHisOc  =  2C2H6O  + 
2CO2,  yielding  (ethyl)  alcohol  and  carbonic  anhydride.  Higher 
alcohols  of  the  fatty  series  are  found  in  traces,  as  also  are  gly- 
cerin, succinic  acid,  and  probably  many  other  bodies.  The  fer- 
mentation is  most  active  at  about  25 °C.  Below  5°C.  or  above 
45° 0.  it  almost  entirely  ceases.  If  the  saccharine  solution  con- 
tains more  than  15  per  cent,  of  sugar  it  will  not  all  be  decom- 
posed, as  excess  of  alcohol  stops  the  reaction.  (2)  Lactic.  This 
is  best  known  as  occurring  in  milk  when  it  turns  sour  owing  to 
the  conversion  of  lactose  into  lactic  acid.  But  dextrose  and 
other  sugars  may  also  be  converted  into  lactic  acid  (CeHioOe  = 
2C3H6O3),  the  conversion  being  ordinarily  due  to  the  presence 
of  some  specific  micro-organism  ^  which  is  specially  active  in 
presence  of  decomposing  nitrogenous  material,  such  as  decaying 
cheese.^  A  similar  change  is  rapidly  produced  when  dextrose  is 
mixed  with  finely  divided  gastric  mucous  membrane.^  There  is 
also  some  evidence  of  the  existence  of  an  unorganised  ferment 
(enzyme)  in  the  mucous  membrane  of  the  stomach  which  can 
convert  lactose  and  dextrose  (?)  into  lactic  acid.*  On  prolonged 
standing  the  lactic  fermentation  is  apt  to  pass  into  (3)  Butyric. 
This  results  from  the  appearance  and  action  of  another  specific 
organised  ferment  on  the  first  formed  lactic  acid,  the  change 
being  accompanied  by  the  evolution  of  hydrogen  and  carbonic 
anhydride  — 

2C3H6O3  =  C3H7.  COOH.  +  2C0o  +  2H2. 

Lactic  and  butyric  fermentations  are  most  active  at  35°  and  40° 
respectively ;  they  probably  occur  constantly  in  the  alimentary 
canal  with  a  carbohydrate  diet  and  may  in  some  cases  be  remark- 
ably predominant.  The  hydrogen  evolved  during  butyric  fermen- 
tation probably  plays  some  important  part  in  the  production  of 
the  f cecal  and  urinary  pigments  from  those  of  bile  (see  below). 

Dextrose  is  the  sugar  which  is  characteristically  formed  by  the 
action  of  boiling  dilute  mineral  acids  on  sugars  of  the  cane-sugar 
group,  and  on  starch  and  dextrin.  When  it  is  dissolved  in  concen- 
trated sulphuric  acid  it  is  said  to  be  partly  reconverted  into  a  true 

1  Lister,  Path.  Soc.  Trans.  1873,  p.  425.  Quart.  JI.  Micros.  Sci.  Vol.  xviii.  (1878), 
p.  177.  Marpmann,  Centralb.  f.  allg.  Gesundheitspfl,  Erganzungshft.  ii.  (1886),  S.  117. 
Meyer,  Inaug.-Diss.  Dorpat,  1880.     Abst.  in  Maly's  Bericht.  1881,  S.  468. 

'■2  Benschj  Preparation  of  lactic  acid.     Liebig's  Ann.  Bd.  lxi.  (1847),  S.  174. 

3  Malv,  Liebig's  Ann.  Bd.  clxxiii.  (1874),  S.  227. 

*  Harnmarsten  (Swedish).     See  Abst.  in  Maly's  Ber.  Bd.  ii.  (1872),  S.  118. 


106  L^VULOSE.     GALACTOSE. 

dextrin  which  may  be  precipitated  by  the  addition  of  alcohol,  and  is 
capable  of  reconversion  into  dextrose  by  mineral  acids. ^ 

2.     Lcevulose. 

CeHioOe.     [CH2.  OH  —  CO  —  (OH.  0H)3  —  CH^.  OH], 

This  is  the  ketone  corresponding  to  the  aldehyde  dextrose.  It 
is  best  known  as  occurring  mixed  with  dextrose  in  many  fruits, 
also  in  honey,  and  is  stated  to  occur  occasionally  in  urine.  It  is 
a  characteristic  product  of  the  action  of  dilute  mineral  acids  on 
cane-sugar,  which  is  hereby  decomposed  into  equal  parts  of  dex- 
trose and  Isevulose,  and  since  when  the  change  is  complete  the 
original  dextro-rotatory  power  of  the  solution  has  become  Isevo- 
rotatory,  the  cane-sugar  is  said  to  have  been  '  inverted.'  A  simi- 
lar inversion  takes  place  in  the  stomach  and  small  intestine  (see 
under  cane-sugar).  In  its  general  reactions  Isevulose  behaves  like 
dextrose,  but  may  be  at  once  distinguished  from  the  latter  by  its 
powerful  Isevo-rotatory  action  on  polarised  light :  this  varies  con- 
siderably with  the  temperature  and  concentration  of  the  solution. 
It  yields  with  phenyl-hydrazin  an  osazone  identical  with  that 
derived  from  dextrose.  It  forms  a  compound  with  calcium  hydrate 
which  unlike  that  yielded  by  dextrose  is  extremely  insoluble  and 
may  thus  be  employed  for  the  separation  of  the  two  sugars. 

2o    Galactose  (Cerebrose)  O^H-uOe- 

When  milk  sugar  (lactose),  see  p,  113,  is  boiled  with  dilute 
mineral  acids  it  is  decomposed  into  a  molecule  of  dextrose  and 
one  of  galactose 

Ciall, Ai -h  H,0  =  CsHi.Oe  +  CsHiA. 

The  two  sugars  may  be  separated  by  crystallisation  and  by  taking 
advantage  of  the  greater  solubility  of  galactose  in  absolute  alcohol.''^ 
In  its  general  reactions  and  behaviour  galactose  resembles  dextrose 
but  is  possessed  of  a  considerably  greater  specific  rotatory  power 
[(a)jj  =-|- 83°]  which  increases  with  the  concentration  and  rise 
of  temperature.^  It  yields  with  phenyl-hydrazin  an  osazone 
(phenyl-galactosazone)  which  has  the  same  composition  as  phenyl- 
glucosazone  and  very  similar  solubilities.  It  differs  however  from 
the  latter  in  melting  at  190 — 193°  and  in  being  optically  inactive 
when  dissolved  in  glacial  acetic  acid.  It  has  recently  been 
shown  that  the  sugar  which  was  described  by  Thudichum*  as 
resulting  from  the  action  of  boiling  dilute  sulphuric  acid  on  cer- 

1  Musculus  u.  Meyer,  Zl  f.  physiol.  Chem.  Bd.  v.  (1881),  S.  122, 

2  Fudakowski,  Ber.  d.  d'.  chem.  Gesell.  Jahrg.  1875,  S.  599,  Soxhlet,  Jn.  f.  pr. 
Chem.   (2)  Bd.  xxi.  (1880),  S.  269. 

3  Meissl,  Jn.  f.  pr.  Chem.  Bd.  xxii.  (1880),  S.  97. 

4  Jn.f.pr.  Chem.  Bd.  xxv.  (1882),  S.  19. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        loV\ 

tain   constituents  of   the   brain    substance,  and  was    named   by 
him  cerebrose,  is  really  identical  with  galactose.^ 

Galactose  is  fermentible  with  yeast,  but  less  readily  so  than  is 
dextrose. 

4.    Glycuronic  acid.    CeHioOv     [COH  -  (CH .  OH)^  -  COOH]. 

This  acid  was  lirst  obtained  as  a  compound,  campho-glycuronic 
acid,  in  the  urine  of  dogs  after  the  administration  of  camphor,^ 
and  subsequently  as  urochloralic  acid  after  the  administration 
of  chloral.^  Since  then  it  has  been  found  in  urine  as  ethereal  or 
glucose-like  compounds,  with  an  extensive  series  of  members  of 
the  fatty  or  aromatic  series  after  the  introduction  of  the  ap- 
propriate substances  into  the  animal  body.*  It  is  probable 
that  traces  of  compounds  of  this  acid  occur  normally  in  urine, 
since  this  excretion  is  usually  slightly  Isevo-rotatory,  and  it  is 
known  that  indol  and  skatol  which  are  formed  in  the  alimentary 
canal  readily  reappear  in  the  urine  as  compounds  of  glycu- 
ronic acid ;  viz.  indoxyl-  and  skatoxyl-glycuronic  acid,  when  intro- 
duced into  the  body.  The  compounds  of  glycuronic  acid  are 
all  leevo-rotatory,  and  some  of  them  reduce  metallic  salts  on 
boiling,  and  may  hence  lead  to  errors  in  the  determination  of 
sugar  in  urine. 

Glycuronic  acid  does  not  occur  in  the  free  state  in  the  animal 
body.  Chemically  it  is  closely  related  to  dextrose;  when  oxi- 
dised with  bromine  it  yields  saccharic  acid,^  CeHioOg,  [COOH  - 
(CH .  0H)4  -  COOH],  —  an  acid  which  is  also  readily  obtained  by 
the  oxidation  of  dextrose  with  nitric  acid.  Saccharic  acid  can  be 
converted  into  glycuronic  acid  by  reduction  with  sodium  amal- 
gam.^ Like  dextrose,  glycuronic  acid  is  dextro-rotatory,  but  to  a 
less  extent,  (a)D  =  -|-  194°,  reduces  Fehling's  fluid  to  the  same 
extent  as  does  dextrose,  and  forms  with  phenyl-hydrazin  a  yellow 
crystalline  compound  which  melts  at  114 — 115°.  The  acid  is 
known  only  as  a  syrup  soluble  in  alcohol  and  water.  When 
boiled  in  the  latter  solvent  it  loses  a  molecule  of  water  and  yields 
an  anhydride  (lactone),  CeHgOe,  which  is  crystalline,  insoluble  in 
alcohol,  soluble  in  water,  dextro-rotatory,  and  reduces  Fehling's 
fluid  powerfully. 

1  Thierfelder,  Zt.  f.  phi/siol.  Cliem.  Bd.  xiv.  (1889),  S.  209.  Browu  and  Morris, 
Jl.  Chem.  Soc.  Vol.  lvh.  (1890),  p.  57. 

'^  Schmiedeberg  u.  Meyer,  Zt.  f.  physiol.  Chem.  Bd.  in.  (1879),  S.  422. 

3  V.  Mering,  Ibid.  Bd.  vi.  (1882),  S'.  480. 

*  For  very  full  list  of  the  various  substances  which  when  introduced  into  the  body 
reappear  in  the  urine  as  paired  compounds  with  glycuronic  acid,  and  for  references 
to  date  (1890)  to  the  literature  of  the  subject,  see  Neubauer  u.  Vogel,  Harnanati/se, 
Ed.  IX.  1890,  p.  116. 

5  Thierfelder,  Zt.  f.  plujsiol.  Chem.  Bd.  xi.  (1887),  S.  388.  See  also  Bd.  xiii, 
(1889),  S.  27.5. 

"  Fischer  u.  Piloty,  Ber.  d.  d.  chem.  Gesell.  Jahrg,  xxiv.  (1891),  S.  521. 


108  INOSIT. 

The  formation  of  the  compounds  of  glycuronic  acid,  to  which 
attention  has  been  drawn,  is  of  great  and  increasing  interest. 
There  can  be  little  doubt  that  the  acid  has  its  origin  in  the  carbo- 
hydrate (dextrose)  of  the  body,  but  it  is  not  yet  possible  to  ex- 
plain exactly  how  each  particular  compound  arises  after  the 
introduction  of  the  corresponding  substance  into  the  animal 
orsanism.i 


Inosit.     CeHiA  +  2H2O.     [CH.OHje. 

This  substance  has  the  same  percentage  composition  as  a  sugar, 
and  possesses  a  distinctly  sweet  taste ;  in  virtue  of  which  prop- 
erties it  appears  to  have  been  usually  classed  with  the  carbo- 
hydrates. It  does  not,  however,  yield  any  of  the  reactions  most 
typical  of  this  class  of  substances;  for  instance,  it  exerts  no 
rotatory  power  on  polarised  light,  does  not  reduce  metallic  salts, 
does  not  undergo  alcoholic  fermentation,  and  does  not  react  with 
phenyl-hydrazin.  On  account  of  these  peculiarities,  the  view  was 
long  ago  expressed  that  it  is  not  a  carbohydrate  at  all;  and 
this  has  recently  been  shown  to  be  the  case  by  Maquenne,  who 
has  proved  that  it  belongs  really  to  the  benzol  series.^  Struc- 
turally it  may  be  represented  by  a  closed  ring  of  six  CH  .  OH 
groups. 

Inosit  occurs  but  sparingly  in  the  human  body ;  it  was  found 
originally  by  Scherer  ^  in  the  muscles.  Cloetta  showed  its  pres- 
ence in  the  lungs,  kidneys,  spleen,  and  liver,*  and  Mliller  in  the 
brain.^  It  occurs  also  in  diabetic  urine,  and  in  that  of  '  Bright' s 
disease,'  and  is  found  in  abundance  in  the  vegetable  kingdom,  — 
more  especially  in  unripe  beans,  from  which  it  may  be  conven- 
iently prepared.^  It  is  also  found  in  the  urine  after  the  ingestion 
of  an  excess  of  water  into  the  body.'' 

It  is  prepared  from  aqueous  extracts  of  the  mother  tissues  by 
acidulating  with  acetic  acid  and  boiling  to  remove  any  coagulable 
proteids.  The  filtrate  from  these  is  then  precipitated  with  normal 
lead  acetate  and  filtered,  and  the  inosit  is  finally  precipitated  from 
this  filtrate  by  means  of  basic  lead  acetate  in  presence  of  ammonia. 
The  lead  compound  is  decomposed  with  sulphuretted  hydrogen, 
and  after  the  addition  of  alcohol  and  ether  to  the  solution, 
inosit  separates  out  by  crystallisation.^ 

Pure  inosit  forms  large  efflorescent  crystals  (rhombic  tables)  ; 

1  In  the  case  of  camphor  and  chloral  see  Fischer  u.  Piloty,  loc.  cit.  S.  524. 

2  Compt.  Rend.  T.  civ.  (1887),  pp.  225,  297,  1719. 

3  Ann.  d.  Chem.  u.  Pharm.  Bd.  lxxiii.  (1850),  S.  322. 
*  Ibid.  Bd.  xcix.  S.  289. 

5  Ibid.  Bd.  cm.  S.  140. 

6  Vohl,  Ibid.  Bd.  xcix.  (1856),  S.  125 ;  ci.  S.  50. 

7  Kiilz,  Centralb.  f.  d.  med.  Wiss.  1875,  S.  933. 

8  Marme',  Arm,  d.  Ch.  u.  Pharm.  Bd.  cxxix.  S.  222.  See  also  Boedeker,  Ihid.  Bd. 
cxvii.  S.  118. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY. 


109 


in  microscopic  preparations  it  is  usually  obtained  in  tufted  lumps 
of  fine  crystals. 


Fig.  1.    Inosit  Crystals.     (After  Kiihue.) 

Eeadily  soluble  in  water,  it  is  only  slightly  so  in  dilute  alcohol, 
and  is  insoluble  in  absolute  alcohol  and  ether. 

Although  inosit  admits  of  no  direct  alcoholic  fermentation,  it 
has  been  stated  to  be  capable  of  undergoing  a  lactic  fermentation 
in  presence  of  decomposing  proteid  (cheese)  and  chalk,  yield- 
ing ordinary  (ethylidene-)  lactic  acid  and  some  butyric  acid.^ 
It  had  been  previously  stated  that  the  acid  thus  obtained  is 
sarcolactic  (ethylene-  or  para-)  lactic  acid.^  These  assertions  are 
scarcely  reconcilable  with  our  present  knowledge  of  the  chemical 
constitution  of  inosit. 


Reactions  of  inosit. 

(i)  Scherers  test.^  The  suspected  substance  is  treated  with 
strong  nitric  acid  and  evaporated  nearly  to  dryness  on  porcelain. 
On  the  addition  of  a  little  ammonia  and  a  few  drops  of  freshly 
prepared  and  not  too  dilute  solution  of  calcium  chloride,  a  bright 
pink  or  rose-coloured  residue  is  obtained  on  renewed  evaporation 
if  inosit  is  present. 

(ii)  Gallois'  test.  When  inosit  in  concentrated  solution  is 
treated  with  a  few  drops  of  2  p.c.  mercuric  nitrate  solution,  or 
Liebig's  solution  for  the  estimation  of  urea,  and  the  mixture  is 
evaporated  to  dryness,  it  yields  a  yellow  residue  which  on  being 
more  strongly  heated  turns  rosy  red ;  this  disappears  on  cooling, 
and  returns  again  on  renewed  heating.* 

1  Vohl,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  1876,  S.  984. 

2  Hilger,  Ann.  d.  Chem.  u.  Pharm.  Bd.  clx.  (1871),  S.  333. 

3  Ann.  d.  Chem.  u.  Pharm.  Bd.  lxxxi.  (1852),  S.  375. 
*  Zt.f.  anal.  Chem.  Bd.  iv.  (1865),  S.  264. 


110  CANE-SUGAR. 

(iii)  SeideVs  reaction}  A  small  amount  (say  -03  gr.)  of  the 
suspected  substance  is  evaporated  to  dryness  in  a  platinum  cruci- 
ble with  a  little  nitric  acid  (sp.  gr.  1*1 — 1-2),  and  the  residue  is 
treated  with  ammonia  and  a  few  drops  of  a  solution  of  strontium 
acetate.  If  inosit  is  present,  a  greenish  colouration  is  observed, 
together  with  a  violet  precipitate. 

The  Cane-Sugak  Geoup. 

I.     Saccharose.     {Cane-sugar.)     C12H22O11. 

Although  it  is  not  found  as  a  constituent  of  any  animal  tissue, 
this  sugar  possesses  no  inconsidera.ble  interest  in  view  of  the  fact 
that  it  is  a  food-stuff  which  is  largely  consumed  by  man,  and 
may  constitute  in  many  cases  no  small  part  of  the  total  carbo- 
hydrates with  which  the  body  is  supplied. 

Cane-sugar  is  chiefly  distinguished  from  the  others  by  the  fact 
that  it  does  not  reduce  metallic  salts,  and  does  not  form  a  com- 
pound with  phenyl-hydrazin ;  but  the  property  which  is  of  great- 
est interest  to  the  physiologist  is  the  ease  with  which  it  may 
be  'inverted'  or  converted  into  equal  parts  of  dextrose  and 
l?evulose :  — 

C12H22OU  -f  H2O  =  CeHioOe  (dextrose)  +  Q.YL^.O,  (Isevulose). 

This  inversion  is  readily  brought  about  by  treatment  with  dilute 
mineral  acids  at  lOO"'-,  or  even  at  40°  or  below  if  the  action  is 
more  prolonged ;  ^  it  is  also  the  result  of  the  action  of  enzymes, 
more  especially  of  invertin  from  yeast,  and  is  characterised  ex- 
perimentally by  the  change  in  the  rotatory  power  of  the  solution, 
which  from  being  originally  dextro-rotatory  becomes  Isevo-rotatory ; 
hence  the  name  '  inversion.'  For  cane-sugar  (a)©  =  +66°  ;  for 
Isevulose  (a)D  =  —100°.  The  rotatory  power  of  the  latter  is 
largely  dependent  upon  temperature  and  concentration. 

A¥hen  cane-sugar  is  injected  into  the  blood-vessels  or  tissues  of 
an  animal  it  is  eliminated  in  an  unaltered  condition,  and  is  thus 
shown  to  be  non-assimilable.^  On  the  other  hand,  it  may  be 
introduced  in  large  amounts  into  the  alimentary  canal  without 
reappearing  externally  in  the  urine.  From  this  it  may  be  con- 
cluded that  it  undergoes  some  change  before  or  during  absorption, 
and  this  change  is  most  probably  that  of  inversion.  This  change 
may  take  place  in  the  stomach,  partly  under  the  influence  of  the 
acid  of  the  gastric  juice,  but  also  as  the  result  of  the  action  of  a 
soluble  enzyme  ;  *  it  is  even  more  marked  in  the  small  intestine, 

1  Dissertation,  Dorpat,  1884.  Quoted  by  Fick.  (Pkarm.  Zt.  f.  Russl.)  See 
Abst.  in  Ber.  d.  d.  chem.  Gesell.  Jahrg.  xx.  (1887),  Ref.  Bd.  S.  320. 

2  Cf.  VV^ohl,  Ibid.  Jalirg.  xxiii.  (1890),  S.  2087. 

^  Bernard,  Lecons  de  Physiol,  exp.  T.  i.  1855,  p.  219. 

*  Leube,   Virchow's  Arch.  Bd.   lxxxviii.    (1882),   S.  222.     Cf.   Hoppe-Seyler, 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.        Ill 

where  the  active  agent  is  without  doubt  an  enzyme.^  From 
this  it  appears  that  cane-sugar  conforms  to  the  apparently 
general  rule  that  the  carbohydrates  leave  the  alimentary  canal 
as  dextrose. 

Cane-sugar  readily  undergoes  a  lactic-acid  fermentation  in  pres- 
ence of  sour  milk  to  which  zinc  oxide  is  added  for  the  fixation  of 
the  acid  as  it  is  formed. 

2.     Maltose.     CioHo.On  +  HoO. 

This  is  the  sugar  which  is  characteristically  formed,  together 
with  dextrins,  by  the  action  of  malt-extract  (diastase)  on  starch- 
paste.  It  was  first  described  by  Dubrunf aut  ^  as  arising  in  this 
way,  but  its  existence  was  for  some  time  doubted  until  firmly 
established  by  O'Sullivan.^  Later  researches  showed  that  it  is 
similarly  the  chief  sugar  which  is  formed  by  the  action  of  saliva 
and  pancreatic  juice  upon  starch-paste  or  upon  glycogen,  being 
accompanied  in  the  case  of  pancreatic  juice  by  a  variable  but  dis- 
tinct amount  of  dextrose  if  the  action  of  this  secretion  be  pro- 
longed.* Maltose  is  also  formed  bv  the  action  of  dilute  acids 
upon  starch-paste,  but  in  this  case  it  is  difficult  to  prevent  the 
simultaneous  formation  of  dextrose  into  which  it  is  readily  con- 
verted by  acids,  yielding  98  —  99  p.  c.  of  the  latter  sugar.^  It  is 
therefore  usually  prepared  from  the  products  of  the  action  of  malt- 
extract  on  starch-paste.*^ 

Maltose  is  very  soluble  in  water,  also  in  alcohol,  but  less  so  in 
the  latter  solvent  than  is  dextrose.  It  crystallises  in  fine  needles 
which  are  however  not  very  easily  obtained.  Solutions  of  maltose 
are  dextro-rotatory  and  reduce  metallic  salts ;  it  is  therefore  not 
easily  distinguished  from  dextrose  by  merely  qualitative  tests. 
As  the  necessity  of  discriminating  between  the  two  sugars  is  one 
of  frequent  occurrence,  the  following  characteristic  differences  be- 
tween their  optical  and  reducing  powers  are  of  great  importance. 
For  maltose  in  10  p.  c.  solution  at  20°C.  (a)D  =  +  140°,'  for  dex- 
trose («.)d=:-|- 52-5°.     When  maltose   is   boiled   with  Fehling's 

Virchow's  Arch.  Bd.  x.  (1856),  S.  144.  Koebner,  Diss,  Breslau,  (1859).  Abst.  in 
Henle  u.  Meissner's  Jahresb.  1859,  S.  236. 

i  Leube,  Centralb.  f.  d.  med.  Wiss.  1868,  S.  289,  Paschutin,  Arch.  f.  Anat.  u. 
Ph)/siol.  Jahrg.  (1871),  S.  374.  Bernard,  Gaz.  med.  de  Paris,  1873,  p.  200.  Brown 
and  Heron,  Liebig's  Ann.  Bd.  cciv.  (1880),  S.  228.  Proc.  Roij.  Soc.  No.  204,  1880, 
p.  393.     Vella,  Moleschott's  Untersuch.  Bd.  xiii.  (1881),  S.  40. 

2  Ann.  Chim.  et  Phi/s.  (3)  T.  xxi.  (1847),  p.  178. 

3  Jl.  Chem.  Soc.  Ser.  2,  Vol.  x.  (1872),  p.  579.  Cf.  Musculus  u.  Gruber,  Zt. 
physiol.  Chem.  Bd.  ii.  (1878-79),  S.  177. 

■*  Musculus  u.  von  Mering,  Zt.f.  phi/siol.  Chem.  Bd.  i.  (1877-78j,  S.  395  ;  ii.  (1878), 
S.  403.  Kiilz,  Pfliiger's  Arch.  Bd.  xxiv.  (1881),  S.  81.  Brown  and  Heron,  Liebig's 
Ann.  Bd.  cxcix.  (1879),  S.  165  ;  Bd.  cciv.  S.  228.  Proc.  Roi/.  Soc.  No.  204,  1880,  p. 
393.     von  Mering,  Zt.f.  physiol.  Chem.  Bd.  v.  (1881),  S.  185. 

5  Meissl,  Jn.  f.  pr.  Chem.  (2),  Bd.  xxv.  (1882),  S.  114. 

6  Soxhlet,  Jii.f,  pr.  Chem.  (2),  Bd.  xxi,  (1880).  Herzfeld,  Liebig's  Ann.  Bd.  ccxx, 
(1884),  S.  211. 

■^  Meissl,  loc.  cit.    Brown  and  Heron  make  it  less  =  +135-4. 


112  MALTOSE. 

fluid  ^  the  amount  of  cuprous  oxide  which  separates  out  is  only 
about  two-thirds  of  that  which  would  be  reduced  by  an  equal 
weight  of  dextrose,  or  in  other  words  66  parts  of  dextrose  reduce 
as  much  as  100  parts  of  maltose.  Bearing  in  mind  that  maltose 
may  be  readily  converted  into  dextrose  by  boiling  with  dilute 
acids  with  a  corresponding  change  of  its  optical  and  reducing 
powers,  while  dextrose  is  of  course  unaltered  by  this  operation,  it 
is  easy  to  base  upon  the  above  facts  a  method  of  identifying  the 
two  sugars.  As  a  further  difference  between  the  two  it  may 
be  stated  that  Barfoed's  reagent  ^  is  not  reduced  by  maltose, 
whereas  it  is  by  dextrose.^  In  this  respect  maltose  resembles 
lactose  (milk-sugar)  which  also  does  not  reduce  this  reagent. 

Fhenyl-maltosazone.    C24H32N4O9. 

This  compound  of  maltose  is  obtained  by  the  action  of  phenyl- 
hydrazin  upon  it  in  presence  of  acetic  acid  in  the  way  already 
described  (p.  104)  for  the  preparation  of  the  analogous  compound 
with  dextrose.  ^  It  crystallises  readily  in  minute  yellow  needles 
and  is  characterised  by  being  (unlike  phenyl-glucosazone)  soluble 
in  about  75  parts  of  boiling  water,  and  still  more  soluble  in  hot 
alcohol.  Its  melting  point  206°  is  practically  the  same  as  that  of 
phenyl-glucosazone. 

The  researches  of  Brown  and  Heron  (see  above,  p.  59)  showed 
that  whereas  pancreatic  juice  rapidly  converts  starch-paste  into 
maltose  and  a  little  dextrose,  an  extract  of  the  mucous  membrane 
of  the  small  intestine  or  the  tissue  itself,  while  acting  but  feebly 
on  starch-paste  rapidly  converts  maltose  into  dextrose.  They 
hence  surmised  that  maltose  would  be  found  to  be  a  non-assimil- 
able sugar,  requiring  like  cane-sugar  to  be  converted  into  the 
simpler  dextrose  before  absorption.  More  recent  experiments  have 
confirmed  this  view,*  for  it  has  been  found  that  if  maltose  be  in- 
jected into  the  blood-vessels  it  is  largely  excreted  in  an  unaltered 
form  in  the  urine.^  The  converting  action  of  extracts  of  the  in- 
testinal mucous  membrane  is  strikingly  less  than  that  of  the' 
tissue  itself ;  from  this  it  may  perhaps  be  inferred  that  the  change 
into  dextrose  takes  place  rather  during  than  previous  to  absorp- 
tion. This  fact  corresponds  closely  to  the  well-known  views  as  to 
the  changes  which  peptones  similarly  undergo  during  their  passage 

1  Solution  of  hydrated  cupric  oxide  in  caustic  soda,  in  presence  of  the  double 
tartrate  of  sodium  and  potassium  (Rochelle  salt).     See  Soxhlet,  loc.  cit. 

^  Dissolve  1  part  of  cupric  acetate  in  15  parts  of  water :  to  200  c.  c.  of  this  solution 
add  5  c.c.  of  acetic  acid  containing  38  p.c.  of  glacial  acid.  Jn.  f.  pr.  Chem.  (2),  Bd.  vi. 
(1872),  S.  334. 

3  Musculus  u.  von  Mering,  loc.  cit. 

*  But  cf.  previously  Bimmermann,  Pfliiger's  Arch.  Bd.  xx.  (1879),  S.  201. 

5  Philips,  Diss.  Amsterdam,  1881.  See  Abst.  in  Maly's  Berickt.  1881,  p.  60.  See 
also  Bourquelot,  Compt.  Rend.  T.  xcvii.  (1883),  pp.  1000,  1322;  T.  xcviii.  p.  1604. 
Journ.  de  I'Anat.  et  de  la  Physiol.  T.  xxii.  (1886),  p.  161. 


CHEMICAL   BASIS   OF  THE  ANIMAL  BODY.        113 

through  the  walls  of  the  intestine  into  the  neighbouring  blood- 
vessels (see  §  309). 

3.     Lactose  (Milk-sugar).     C12H22OU  +  H2O. 

It  is  found  characteristically  and  solely  in  milk,  in  quantities 
varying  with  the  class  of  animal  and  at  different  times  with  the 
same  animal.^  The  percentage  is  relatively  high  in  human  milk. 
It  is  also  said  to  occur  in  the  urine  of  lying-in  women  and 
sucklings.^ 

Preparation.  The  casein  is  precipitated  from  diluted  milk  by 
the  addition  of  acetic  acid.  The  filtrate  from  this  is  boiled  to 
coagulate  the  remaining  proteids,  which  are  then  removed  by  fil- 
tration. This  final  filtrate  is  then  concentrated,  and  on  prolonged 
standing  yields  crusts  of  milk  sugar  which  are  purified  by  recrys- 
tallisation  from  hot  water. 

It  yields,  when  pure,  hard  colourless  crystals,  belonging  to  the 
rhombic  system  (four-sided  prisms).  It  is  less  soluble  in  water 
than  dextrose,  requiring  for  solution  six  times  its  weight  of  cold, 
but  only  two  parts  of  boiling,  water ;  it  is  entirely  insoluble  in 
alcohol  and  in  ether.  It  is  fully  precipitated  from  its  solutions  by 
the  addition  of  basic  lead  acetate  and  ammonia. 

Solutions  of  many  metallic  salts  are  readily  reduced  by  boiling 
with  lactose,  but  the  reducing  power  is  less  than  that  of  dextrose. 
Thus  1  c.  c.  of  Fehling's  fluid  which  is  reduced  by  5  mgr.  of  dex- 
trose requires  6*7  mgr.  of  lactose  provided  that  certain  conditions 
as  to  the  dilution  of  the  solution,  duration  of  boiling,  &c.,  are 
attended  to.''^  These  are  important  for  the  accurate  volumetric 
estimation  of  lactose.  The  specific  rotatory  power  of  lactose  is 
(a)jj  =  -[-52-3°,  and  is  independent  of  the  concentration  in  solu- 
tions which  contain  up  to  35  p.  c.  at  ordinary  temperatures.  Its 
rotatory  power  is  thus  identical  with  that  of  dextrose.  It  is,  how- 
ever, readily  distinguishable  from  dextrose  by  its  smaller  solubility 
in  water,  insolubility  in  alcohol,  and  incapability  of  undergoing 
direct  alcoholic  fermentation  with  yeast.  It  also  does  not  reduce 
Barfoed's  reagent,  and  in  this  resembles  maltose.  When  boiled 
with  dilute  mineral  acids  it  yields  equal  molecules  of  dextrose  and 
galactose  (see  p.  106),  and  since  the  specific  rotatory  power  of  the 
latter  of  these  is  high  [(a)D  = +  83°],  this  increase  of  rotatory 
(and  reducing)  power  on  treatment  with  acids  affords  a  further 
convenient  means  of  discrimination  between  lactose  and  dextrose. 

1  See  Gorup-Besanez,  Lehrb.  d.  phi/siol.  Chem.  1878,  S.  444.  Konig,  Chem.  d.  mensch 
Nahrungs-  u.  Genussmittel,  3  Aufl.  (1889),  Bd.  i.  S.  250  et  seq. 

■■2  Hofmeister,  Zt.  f.  phi/siol.  Chem.  Bd.  i.  (1877),  S.  101.  See  Neubauer  u.  Vogel, 
Analyse  d.  Hams,  2'Theil,  1890,  S.  48. 

3  Rodewald  u.  Tollens,  Ber.  d.  d.  chem.  Gesell.  1878,  S.  2076.  Soxhlet,  Zt.  f. 
prakt.  Chem.  (2),  Bd.  xxi.  1880,  S.  227. 


114  LACTOSE. 


Fhenyl-lactosazonc.     €241132^409. 

This  compound  of  lactose  with  phenyl-hydrazin  is  formed  under 
conditions  similar  to  those  already  described  for  the  preparation 
of  the  analogous  compound  of  dextrose.  It  is  soluble  in  80  —  90 
parts  of  boiling  water  and  melts  at  about  200°.  It  crystallises 
readily  in  the  form  of  yellow  needles  which,  unlike  the  crystals 
of  phenyl-maltosazone,  are  usually  aggregated  into  clusters. 

Lactose  is  readily  capable  of  undergoing  a  direct  lactic  fermen- 
tation and  this  occurs  characteristically  in  souring  milk.  The 
exciting  cause  is  doubtless  ordinarily  an  organised  ferment,  but 
there  is  also  some  evidence  of  the  existence  in  the  alimentary 
canal  of  an  enzyme  which  can  effect  the  same  conversion.  The 
circumstances  and  products  of  the  conversion  are  the  same  as  for 
dextrose  and  saccharose. 

Although  isolated  lactose  is  unaffected  by  j^east,  milk  itself  is  cap- 
able of  undergoing,  under  the  influence  of  certain  ferments,  an 
alcoholic  fermentation,  and  this  has  been  employed  from  very  early 
times  by  the  inhabitants  of  certain  districts  of  Russia  in  the  prepara- 
tion of  Kumys  and  Kephir  from  mare's-milk.  Of  late  years  these 
fluids  have  attracted  much  attention  in  virtue  of  their  supposed  thera- 
peutic action  in  certain  wasting  diseases.  Very  little  is  as  yet  known 
as  to  the  real  nature  of  the  changes  which  occur  during  the  fermenta- 
tion, but  they  are  probably  extremely  complex  and  due  to  the  presence 
of  several  organised  ferments.  ^  Kephir  ferment  is  a  commercial 
article  in  Russia,  obtainable  at  the  apothecaries. 

The  non-assimilability  of  saccharose  and  maltose  has  already 
been  referred  to,  and  experiment  has  shown  that  lactose  is  simi- 
larly incapable  of  assimilation,  for  when  injected  into  the  blood- 
vessels it  appears  unaltered  in  the  urine.^  It  is  therefore 
presumably  changed  in  the  alimentary  canal  into  some  form  of 
sugar  which  is  assimilable,  it  may  be  into  dextrose  and  galactose. 
It  does  not  appear  that  any  such  conversion  can  be  markedly 
observed,  if  at  all,  under  the  action  of  any  of  the  secretions  of  the 
alimentary  canal ;  hence  the  change  may  more  probably  take  place, 
as  in  the  case  of  maltose,  rather  during  than  before  the  passage  of 
the  sugar  through  the  intestinal  walls. 

This  non-assimilability  of  lactose  is  certainly  remarkable  when 
it  is  remembered  that  it  is  in  this  form  that  young  animals  receive 

1  There  is  an  extensive  literature  on  this  subject,  of  which  the  following  are  of 
most  comprehensive  interest.  Biel,  [Inters,  iiber  den  Kioni/s,  Wien,  1874,  and  St. 
Petersburg,  1881.  Abst,  in  Maly's  Bencht.  1874,  p.  166,  1886,  p.  159.  Struve,  Ber. 
d.  d.  chem.  Gesell.  Jahrg.  1884,  Sn.  314,  1364.  Krannhals,  Deutsch.  Arch.  f.  klin.  Med. 
Bd.  XXXV.  (1884),  S.  18.  Hammarsten  (Swedish).  See  Abst.  in  Malv's  Bericht. 
1886,  p.  163. 

2  Dastre,  Compt.  Rend.  T.  xcvi.  (1883),  p.  932.  Compt.  Rend.  Soc.  Biol.  (9),  T.  i. 
(1889),  p.  145.     De  Jong  (Dutch  Diss.).     See  Maly's  Bericht.  1886,  p.  445. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        115 

their  supply  of  carbohydrate  food.  It  might  more  probably  have 
been  expected  that  they  should  be  shielded  as  far  as  possible  from 
any  avoidable  excessive  digestive  labour  by  the  presentation  of  a 
directly  assimilable  sugar.  We  cannot  as  yet  offer  any  other  ex- 
planation of  the  observed  facts  than  the  one  that  since  lactose  is 
incapable  of  direct  (alcoholic)  fermentation,  not  only  is  the  milk 
while  it  is  accumulated  in  the  breast  less  liable  to  fermentative 
decomposition,  but  also  the  tendency  to  fermentative  disturbance 
in  the  alimentary  canal  of  the  young  animal  is  largely  diminished. 
Both  saccharose  (cane-sugar)  and  maltose^  are  similarly  not 
directly  fermentable,  and  both  again  in  the  adult  are  apparently 
converted  into  ferhientable  dextrose  during,  or  at  least,  immedi- 
ately before,  absorption.  The  subject  is  one  which  requires  further 
investigation. 


FATTY  ACIDS  AND  FATS,  THEIR  DERIVATIVES 
AND  ALLIES. 

I.    Acids  of  the  Acetic  Series. 

General  formula  C„H2„+i.C00H  (monobasic). 

This,  which  is  one  of  the  most  complete  homologous  series  of 
organic  chemistry,  runs  parallel  to  the  series  of  monatomic  alco- 
hols. Thus  formic  acid  corresponds  to  methyl  alcohol,  acetic  acid 
to  ethyl  (ordinary)  alcohol,  and  so  on.  The  several  acids  may 
be  regarded  as  being  derived  from  their  respective  alcohols  by 
simple  oxidation  taking  place  in  two  stages,  the  first  yielding  an 
aldehyde,  the  second  an  acid  by  direct  union  of  oxygen  with  the 
aldehyde.2     Thus  with  ethyl  alcohol 

(i)  CHs.CH^.OH  +  O^CHs.COH  +  HoO, 

(ii)  CHs .  COH  +  0  =  CHs .  COOH. 

The  successive  members  differ  in  composition  by  CH2,  and  the 
boiling  points  rise  successively  by  about  19°C.  Similar  rela- 
tions hold  good  with  regard  to  their  melting-points  and  specific 
gravities.  The  acid  properties  are  strongest  in  those  where  n 
has  the  least  value.  The  lowest  members  of  the  series  are  volatile 
liquids,  acting  as  powerful  acids ;  these  successively  become  less 

1  Horace  Brown.  Private  communication  to  author.  Cf.  v.  Mering,  Zt.  f. 
»A;/siW.  CAe?7i.Bd.  V.  (1881),  S   189 

'  2  The  views  as  to  the  possible  importance  of  the  aldehydes  have  already  been 
referred  to  when  treating  of  proteids  (see  p.  52).  It  is  further  interesting  to  notice 
that  a  simple  polymerisation,  to  which  it  is  very  prone,  of  the  lowest  (meth-)  aldehyde 
H  .  COH,  would  yield  a  substance  having  the  composition  of  a  carbohydrate.  This  is 
indeed  a  view  which  is  held  by  many  as  to  the  mode  of  formation  of  starch  in  plants. 
Cf.  Miller's  Chemistry,  Part  lii.  1880,  Sec.  I,  p.  726. 


116  ACIDS   OP  THE  ACETIC   SEEIES. 

and  less  fluid ;  and  the  highest  members  are  colourless  solids, 
closely  resembling  the  neutral  fats  in  outward  appearance.  Con- 
secutive acids  of  the  series  present  but  very  small  differences  of 
chemical  and  physical  properties,  hence  the  difficulty  of  separat- 
ing them :  this  is  further  increased  in  the  animal  body  by  the 
fact  that  exactly  those  acids  which  present  the  greatest  similar- 
ities usually  occur  together,  ^ 

The  free  acids  are  found  only  in  small  and  very  variable  quan- 
tities in  various  parts  of  the  body ;  their  derivatives  on  the  other 
hand  form  most  important  constituents  of  the  human  frame,  and 
will  be  considered  further  on. 

Some  of  the  lower  acids  of  the  series  have  been  obtained  by 
treating  proteids  with  molten  caustic  potash.  They  also  occur 
among  the  products  of  the  putrefaction  of  proteids,  as  for  instance 
in  old  cheese. 

Of  the  primary  alcohols  from  which  this  series  of  acids  is  de- 
rived only  two  have  as  yet  been  obtained  from  animal  tissues  or 
secretions,  viz.  ethyl ''^-  and  cetyl-alcohol,^  C2H5 .  OH  and  C16H33 . 
OH,  —  the  former  from  muscle,  brain,  and  liver,  the  latter  in  union 
with  palmitic  acid  in  spermaceti  and  the  secretion  of  the  caudal 
glands  of  birds. 

Formic  acid.      H  .  COOH. 

When  pure  is  a  strongly  corrosive,  fuming  fluid,  with  power- 
ful irritating  odour,  solidifying  at  0°  C,  boiling  at  100°  C,  and 
capable  of  being  mixed  in  all  proportions  with  either  water 
or  alcohol.  It  has  been  obtained  from  various  parts  of  the 
body,  such  as  the  spleen,  thymus,  pancreas,  muscles,  brain,  and 
blood  ;  in  the  latter  its  presence  may  be  due  to  the  action  of  acids 
on  the  haemoglobin.  It  also  occurs  in  minute  traces  in  urine.  It 
is  excreted  by  some  ants  (Formica  rufa)  in  a  fairly  concentrated 
form  and  may  be  present  to  the  surprisingly  large  extent  of  40  p.c. 
in  the  secretion  of  certain  caterpillars.*  The  separation  of  so  acid 
a  fluid  from  the  alkaline  cell-substance  is  remarkable  and  of  con- 
siderable interest.  When  heated  with  strong  sulphuric  acid  it  is 
decomposed  into  carbonic  oxide  and  water.  It  is  further  charac- 
terised by  readily  effecting  the  reduction  of  metallic  salts,  as  of 
mercury  or  silver,  when  heated  with  their  solutions. 

Acetic  Acid.     CH3 .  COOH. 

It  is  distinguished  by  its  characteristic  odour ;  its  boiling-point 
is  100°  C. ;  the  anhydrous  acid  solidifies  at  about  17°.  It  is  solu- 
ble in  all  proportions  in  alcohol  and  in  water. 

1  For  details  on  this  series  see  Hoppe-Seyler's  Hdbch.  d.  phys.  path.  chem.  Anal. 
1883,  S.  85  et  seq. 

2  Rajewski,  Pfliiger's  Arch.  Bd.  xi.  (1875),  S.  122. 

a  De  Jonge,  Zt.f.  physiol.  Chem.  Bd.  iii.  (1879),  S.  225. 

*  Poulton,  The  colours  of  animals,  Internat.  Sci.  Ser,  1890,  p.  274. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY,        117 

It  may  be  formed  in  the  stomach  as  the  result  of  fermentative 
changes  in  the  food,  and  is  frequently  present  in  diabetic  urine,  as 
also  in  traces  in  normal  urine.  In  other  organs  and  fluids  it  exists 
only  in  minute  traces. 

With  ferric  chloride  it  yields  a  blood-red  solution,  decolourised  by 
hydrocloric  acid.  (It  differs  in  this  last  reaction  from  sulphocyanide 
of  iron.)  Heated  with  alcohol  and  sulphuric  acid,  the  characteristic 
odour  of  acetic  ether  (ethyl-acetate)  is  obtained. 

Acetone.     CH3 .  CO  .  CH3. 

This  substance  is  the  typical  member  of  the  general  class  known 
as  ketones,  and  may  be  prepared  by  the  dry  distillation  of  calcium 
or  barium  acetate. 

Ketones  are  characterised  by  containing  the  group  CO  (carbonyl)  in 
the  same  way  that  the  aldehydes  are  characterised  by  the  group  COH, 
and  the  acids  by  the  group  COOH.  The  ketones  are  closely  related 
to  the  aldehydes  and  may  be  regarded  as  derived  from  them  by  dis- 
placing the  H  of  the  COH  group  by  some  monad  (alcohol)  radicle. 
They  are  most  usually  prepared  by  the  dry  distillation  of  the  calcium 
salts  of  the  appropriate  acids.  Ketones,  like  the  aldehydes,  unite 
readily  and  directly  with  phenyl -hydrazin,  yielding  a  class  of  com- 
pounds,  known  as  hydrazones.      (Cf.  p.  102.) 

Acetone  is  a  volatile  liquid,  soluble  in  water,  boiling  at  56°,  and 
possessed  of  an  agreeable  ethereal  odour.  It  may  be  obtained  in 
considerable  quantity  by  distillation  from  the  urine  and  blood  of 
diabetic  patients  and  accounts  for  the  peculiar  ethereal  odour  which 
these  frequently  evolve.^  This  symptom  is  of  serious  prognostic 
importance,  and  it  has  been  supposed  by  many  authors  that  the 
fatal  diabetic  coma  which  rapidly  supervenes  is  caused  by  the  pres- 
ence of  acetone.^  The  urine  of  diabetic  patients  frequently  ex- 
hibits a  reddish-violet  colouration  with  ferric  chloride,  supposedly 
due  to  the  presence  of  aceto-acetic  acid  (CH3 .  CO .  CH2 .  C!OOH) 
which  readily  yields  acetone  by  its  decomposition. 

Acetone  is  also  not  infrequently  'found  in  the  urine  and 
breath  (?)  of  children  in  apparently  normal  health.^ 

Acetone  gives  a  characteristic  reaction  with  iodine  in  presence 
of  an  alkali  (formation  of  iodoform)  and  colour-reactions  with 
sodium  nitro-prusside  and  fuchsin.* 

Propionic  acid.    C0H5 .  COOH. 

This  acid  closely  resembles  the  preceding  one.  It  possesses 
a  very  sour  taste  and  pungent  odour ;  is  soluble  in  water,  boils 

1  Von  Jaksch,  Ueher  Acetonnrie  ti.  Diaceturie,  Berlin,  1885.  Gives  history  aud 
literature  of  the  subject.     Cf.  Zt.  f.  physiol.  Chem.  Bd.  vi.  (1882),  S.  541. 

2  Cf.  Gamgee's  Physiol.  Chem.  Vol.  i.  1880.  p.  168. 

3  Baginsky,  Arch.  f.  Phi/siol.  Jahrg.  1887,  S.  349. 

*  Consult  Neubauer  und  Vogel,  Ilarrnmalijse,  S.  31. 


118  ACIDS   OF   THE   ACETIC   SERIES. 

at  141°  C,  and  may  be  separated  from  formic  and  acetic  acid 
by  taking  advantage  of  the  superior  solubility  of  its  lead  salt 
in  cold  water. 

It  occurs  in  small  quantities  in  sweat,  in  the  contents  of  the 
stomach,  and  in  diabetic  urine  when  undergoing  fermentation. 
It  is  similarly  produced,  mixed  however  with  other  products, 
during  alcoholic  fermentation. 

It  is  stated  to  have  been  found  occasionally  in  normal  urine. 

Butyric  acid.    C3H7 .  COOH. 

There  are  two  possible  isomeric  acids  of  the  general  formula 
C3H7 .  COOH,  the  normal  or  primary,  CII3 .  CH2 .  CHg .  COOH  and 
iso-  or  secondary,  CH(CH3)2 .  COOH. 

Normal  hutyric  add.  An  oily  colourless  liquid,  with  an  odour 
of  rancid  butter,  soluble  in  water,  alcohol,  and  ether,  boiling  at 
162°  C. 

Found  in  sweat,  the  contents  of  the  large  intestine,  faeces,  and 
in  urine.  It  occurs  in  traces  in  many  other  fluids,  and  is  plenti- 
fully obtained  when  diabetic  urine  is  mixed  with  powdered  chalk 
and  kept  at  a  temperature  of  35°  C.  It  exists,  in  union  with  gly- 
cerin as  a  neutral  fat,  in  small  quantities  in  milk,  and  gives  the 
characteristic  odour  to  butter  which  has  become  rancid. 

It  is  the  principal  product  of  the  second  stage  of  lactic  fermen- 
tation (see  p.  105),  and  is  ordinarily  prepared  from  this  source. 

Isohutyric  acid.  Occurs  in  faeces  and  among  the  putrefactive 
products  from  proteids,  also  in  certain  fruits  such  as  the  banana. 

Valeric  or  Valerianic  acid.     C4H9 .  COOH. 

Four  isomeric  forms  of  this  acid  exist.  Of  these  the  one  here 
described  is  the  isoprimary  CH(CH3)2CH2.  COOH.  (Isopropyl- 
acetic  acid.) 

An  oily  liquid,  of  burning  taste  and  penetrating  odour  as  of  de- 
caying cheese  ;  soluble  in  30  parts  of  water  at  12°C.,  readily  soluble 
in  alcohol  and  in  ether.     Boils  at  175°  C. 

It  is  found  in  the  solid  excrements,  and  is  formed  readily  by 
the  decomposition,  through  putrefaction,  of  impure  leucin,  am- 
monia being  at  the  same  time  evolved  ;  hence  its  occurrence  in 
urine  when  that  fluid  contains  leucin,  as  in  cases  of  acute  atrophy 
of  the  liver. 

Caproic  acid.     C5H11 .  COOH. 

Caprylic  acid.     C7H15  .  COOH. 
Capric  (Eutic)  acid.    C9H19 .  COOH. 

These  three  occur  together  (as  fats)  in  butter,  and  are  con- 
tained in  varying  proportions  in  the  faeces  from  a  meat  diet  and 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        119 

the  first  two  in  sweat.  The  first  is  an  oily  fluid,  slightly  soluble 
in  water,  the  others  are  solids  and  scarcely  soluble  in  water ;  they 
are  soluble  in  all  proportions  in  alcohol  and  in  ether.  They  may 
be  prepared  from  butter,  and  separated  by  the  varying  solubilities 
of  their  barium  salts. 

Laurie  or  Laurostearic  acid.    GiJI^s  •  COOH. 
Myristic  acid.    C13H27  .  COOH. 

These  occur  as  neutral  fats  in  spermaceti,  in  butter  and  other 
fats.     They  present  no  points  of  interest. 

Palmitic  acid.     C15H31 .  COOH. 
Stearic  acid.    C17H35 .  COOH. 

These  are  solid,  colourless  when  pure,  tasteless,  odourless,  crys- 
talline bodies,  the  former  melting  at  62°  C,  the  latter  at  69*2°  C. 
In  water  they  are  quite  insoluble ;  palmitic  acid  is  more  readily 
soluble  in  cold  alcohol  than  stearic  :  both  are  readily  dissolved 
by  hot  alcohol,  ether,  or  chloroform.  Glacial  acetic  acid  dissolves 
them  in  large  quantity,  the  solution  being  assisted  by  warming. 
They  readily  form  soaps  with  the  alkalis,  also  with  many  other 
metals.  The  varying  solubilities  of  their  barium  salts  afford  the 
means  of  separating  them  when  mixed :  ^  this  method  may  also 
be  applied  to  many  others  of  the  higher  members  of  this  series. 

These  acids  in  combination  with  glycerin  (see  below),  together 
with  the  analogous  compound  of  oleic  acid,  form  the  principal 
constituents  of  human  fat.  As  salts  of  calcium  they  occur  in  the 
faeces  and  in  '  adipocire,'  and  probably  in  chyle,  blood,  and  serous 
fluids,  as  salts  of  sodium.  They  are  found  in  the  free  state  in 
decomposing  pus,  and  in  the  caseous  deposits  of  tuberculosis. 

The  existence  of  margaric  acid,  as  obtained  from  natural  fats,  in- 
termediate to  the  above  two,  is  not  now  admitted,  since  Heintz  has 
shown  ^  that  it  is  really  a  mixture  of  palmitic  and  stearic  acids. 
Margaric  acid  possesses  the  anomalous  melting-point  of  59 '9°  C.  A 
mixture  of  60  parts  stearic  acid  and  40  of  palmitic  acid,  melts  at  60-3°. 
A  true  margaric  acid  may  however  be  prepared  by  replacing  the  group 
OH  in  cetyl-alcohol  (CisHgs  •  OH)  by  the  group  COOH. 

Adipocire.  When  animal  (proteid)  tissues  are  buried  for  some 
time  in  damp  ground  or  otherwise  exposed  to  moisture  in  the 
absence  of  any  free  supply  of  oxygen  they  are  frequently  found 
to  have  undergone  a  peculiar  change  by  which  they  are  converted 
into  a  waxy  or  fatty  substance.  This  is  known  as  adipocire.  It 
consists,  not  of  true  neutral  fats,  but  of  the  ammonium,  and  in 
some  cases  calcium,  salts  of  the  highest  fatty  acids  palmitic  and 

^  Heintz,  Poggendorff's  Annul,  d.  Phys.  u.  Chem.  Bd.  xcii.  S.  588. 
^  Op.  at. 


120  OLEIC  ACID.     NEUTRAL  EATS. 

stearic,  or  of  the  free  acids  themselves.^  Practically  nothing  is 
definitely  known  as  to  the  agencies  and  mode  of  this  conversion. 
It  may  be  the  result  of  a  purely  chemical  change,  or  perhaps  it  is 
more  probably  due  to  the  action  of  some  micro-organism.^  On 
either  view  of  its  formation  the  occurrence  of  adipocire  is  of 
extreme  interest  as  showing  a  possible  direct  formation  of  the 
higher  fatty  acids  and  hence  of  fats  from  proteids.  It  is  however 
supposed  by  some  authors  that  the  adipocire  is  formed  entirely 
by  change  and  aggregation  from  the  fats  present  in  the  tissues  at 
death.^     This  view  is  probably  incorrect. 

II.    Acids  of  the  Oleic  (Acrylic)  Series.    C„H2;j_i  .  COOH 

(monobasic). 

The  acids  of  this  series  bear  the  same  relationship  to  the  de- 
fines (C2II4)  that  those  of  the  acetic  do  to  the  paraffins  (CH4). 
Some  of  the  higher  members  of  the  series  are  found  as  glycerin 
compounds  in  various  fats. 

They  bear  an  interesting  relation  to  the  acids  of  the  acetic  series, 
breaking  up  when  heated  with  caustic  potash  into  acetic  acid  and 
some  other  member  of  the  same  series  :  —  thus. 

Oleic  acid.  Potassium  acetate.-     Potassium  palraitate. 

Ci7H33.COOH  +  2KHO=    KC2H3O2     +    KCieHaiO^+K.. 

Oleic  acid.    CnHgg .  COOH. 

This  is  the  only  acid  of  the  series  which  is  physiologically  im- 
portant. It  is  found  united  with  glycerin  in  all  the  fats  of  the 
human  body. 

When  pure  it  is,  at  ordinary  temperatures,  a  colourless,  odour- 
less, tasteless,  oily  liquid,  solidifying  at  4°  C.  to  a  crystalline 
mass.  Insoluble  in  water,  it  is  soluljle  in  alcohol  and  in  ether. 
It  cannot  be  distilled  without  decomposition.  It  readily  forms 
with  potassium  and  sodium  hydroxide  soaps  which  are  soluble  in 
water :  its  compounds  with  most  other  bases  are  insoluble.  It 
may  be  distinguished  from  the  acids  of  the  acetic  series  by  its 
reaction  with  nitrous  acid  which  converts  it  into  a  solid  (elaidic 
acid)  and  by  the  changes  it  undergoes  when  exposed  to  the  air. 
It  may  be  converted  into  stearic  acid 

Cx,H33 .  COOH  -f  H2  ==  Ci,H35 .  COOH. 

The  Neutral  Fats. 

These  may  be  considered  as  ethereal  salts  formed  by  replacing 
the  exchangeable    atoms  of   hydrogen  in   the  triatomic  alcohol 

1  Ebert,  Ber.  d.  d.  chem.  Gesell.  Bd.  viii.  (1875),  S.  775. 

2  Kratter,  Zt.  f.  Biol.  Bd.  xvi.  (1880),  S.  455.  Lehmann,  Sitzh.  d.  phus.-med. 
Gesell.  Wiirzburg.  1888,  S.  19. 

3  Zillner,  Viertelj.f.ger.  Med.  u.  off.  Sanitdtsw.  (IST-F.)  Bd.  xnv.  (1885),  S.  1. 


CHEMICAL  BASIS   OF   THE  AXIMAL  BODY.        121 

glycerin  (see  below),  by  the  acid  radicles  of  the  acetic  and  oleic 
series.  Since  there  are  three  such  exchangeable  atoms  of  hydro- 
gen in  glycerin,  it  is  possible  to  form  three  classes  of  these  ethe- 
real salts  ;  only  those,  however,  which  belong  to  the  third  class 
occur  as  natural  constituents  of  the  human  body  :  those  of  the 
first  and  second  are  of  theoretical  importance  only. 

The  following  reaction  which  represents  the  formation  of  tri- 
palmitin  from  glycerin  and  palmitic  acid  is  typical  for  all  the 
others. 

Glj'cerin.  Palmitic  acid.  Tri-palmitin. 

C3H5  (0Hj3  -f  3  (C15H31 .  CO .  OH)  =  C3H5  (C15H31 .  CO .  0)3+  3  HoO. 

They  possess  certain  general  characteristics.  Insoluble  in  water 
and  but  slightly  in  alcohol,  they  are  readily  soluble  in  ether, 
chloroform,  benzol,  &c. ;  they  also  dissolve  one  another.  They 
are  neutral  bodies,  colourless  and  tasteless  when  pure ;  they  are 
not  capable  of  being  distilled  without  undergoing  decomposition, 
and  yield  as  a  result  of  this  decomposition  solid  and  liquid  hydro- 
carbons, water,  fatty  acids,  and  a  peculiar  substance,  acrolein, 
resulting  from  the  decomposition  of  the  glycerin.     (See  below.) 

They  possess  no  action  on  polarised  light. 

They  may  readily  be  decomposed  into  glycerin  and  their  respec- 
tive fatty  acids  by  the  action  of  caustic  alkalis,  or  of  superheated 
steam. 

Palmitin  (Tri-palmitin).    C3H5  (C15H31 .  CO  .  0)3. 

Palmitin  is  but  slightly  soluble  in  alcohol  either  cold  or  hot, 
readily  so  in  ether,  from  which,  when  pure,  it  crystallises  in  fine 
needles ;  if  mixed  with  stearin  it  generally  forms  shapeless  lumps, 
although  the  mixture  may  at  times  assume  a  crystalline  form, 
and  was  then  regarded  as  a  distinct  body,  namely  margarin. 
When  pure  it  melts  at  62°  and  solidifies  again  at  45°. 

It  is  most  conveniently  obtained  from  palm-oil  by  removing  the 
free  palmitic  and  oleic  acids  by  alcohol  and  repeatedly  crystallising 
the  residue  from  ether. 

Stearin  (Tri-stearin).  C3H5  (C17H35 .  CO  .  0)3. 

This  is  the  hardest  and  least  fusible  of  the  ordinary  fats  of  the 
body ;  is  also  the  least  soluble,  and  hence  is  the  first  to  crystallise 
out  from  solutions  of  the  mixed  fats.  Eeadily  soluble  in  ether 
and  in  boiling  alcohol.  It  crystallises  usually  in  square  tables 
or  glittering  plates.  It  presents  peculiarities  in  its  fusing-points, 
melting  first  at  55°,  then  solidifying  as  the  temperature  is  further 
raised,  and  melting  finally  and  permanently  at  71°. 

Preparation.  From  mutton  suet,  its  separation  from  palmitin 
and  olein  being  effected  by  repeated  crystallisation  from  ether, 
stearin  being  the  least  soluble.  It  is,  however,  very  difficult  to 
obtain  it  pure  by  this  process. 


]i22  NEUTEAL  FATS. 

Olein    (Tri-olein).     C3H5  (Ci^H^.s .  C0.0)3. 

Is  obtained  with  difficulty  in  the  pure  state,  and  is  then  fluid 
at  ordinary  temperatures.  It  is  somewhat  soluble  in  alcohol,  very 
soluble  in  ether.  It  readily  undergoes  oxidation  when  exposed  to 
the  air,  and  is  converted  by  mere  traces  of  nitrous  acid  into  a 
solid  isomeric  fat,  tri-elaidin.  Olein  is  saponified  with  much 
greater  difficulty  than  are  palmitin  and  stearin. 

Preparation.  From  olive  oil,  either  by  cooling  to  0°  C.  and 
pressing  out  the  olein  that  remains  fluid,  or  by  dissolving  in  hot 
alcohol  and  cooling,  when  the  olein  remains  in  solution  while  the 
other  fats  crystallise  out. 

The  fats  which  occur  in  the  animal  body  are  mixtures  of  the 
above  three  substances  in  varying  proportions.  The  normal  fat  of 
each  animal  or  class  of  animals  is  however  characterised  by  the 
constant  preponderance  of  one  of  the  three ;  thus  in  the  fat  of 
man  and  carnivora  palmitin  is  in  excess  over  the  other  two.  In 
the  fat  of  herbivora  stearin  predominates,  and  in  that  of  fishes 
olein.  Butter  contains,  in  addition  to  the  above,  several  fats 
formed  by  the  union  of  glycerin  with  the  radicles  of  the  lower 
acids  of  the  acetic  series. 

There  is  no  doubt  that  a  large  -part  of  the  fat  laid  on  in  the 
animal  body  during  fattening  cannot  be  accounted  for  by  the  fat 
given  in  the  food,  and  must  hence  arise  from  a  conversion  of  proteids 
or  carbohydrates  into  fat.  (See  §§  506,  507.)  The  question  as  to 
lioiu  the  storage  arises  from  these  food-stuffs  is  one  which  has 
given  rise  to  a  prolonged  controversy.  On  the  one  hand  Voit 
and  his  followers  urged  that  although  carbohydrates  do  lead  to  a 
rapid  storing  of  fat  in  the  body,  they  do  so  not  directly  by  being 
themselves  converted  into  fat,  but  indirectly  by  protecting  the 
proteids  from  the  metabolism  they  would  otherwise  have  under- 
gone. According  to  this  view  fat  is  formed  from  proteids  only. 
Lawes  and  G-ilbert  on  the  other  hand  took  the  view  that  carbo- 
hydrates are  directly  converted  into  fat.  While  there  is  no  doubt 
that  proteids  can  give  rise  directly  to  fat  as  shown  by  the  storage 
of  fat  during  "nitrogenous  equilibrium"  (see  §  522),  there  is 
also  now  equally  no  doubt  that  carbohydrates  can  lead  to  a  direct 
storage  of  fat  by  being  themselves  converted  into  fat.  This  is 
the  incontrovertible  outcome  of  the  most  recent  experiments, 
which  have  proved  that  with  a  diet  rich  in  carbohydrates,  so  that 
the  storage  of  fat  is  sufficiently  rapid,  more  fat  is  laid  on  than 
could  possibly  have  been  formed  from  the  proteids  in  the  food 
given.^ 

1  Meissl  u.  Strohmer,  Sitzb.  d.  Wien.  Ahad.  Bd.  Lxxxvin.  1883,  III.  Abtli.  July. 
Tscherwinskv,  Landwirth.  Versuchsstat.  Bd.  xxix.  (1883),  S.  317.  -  Chaniewski,  Zif. 
f.  Biol.  Bd.'xx.  (1884),  S.  179.  Rubner,  Ibid.  Bd.  xxii.  (1886),  S.  272.  Munk, 
Vircnow's  Arch.  Bd.  ci.  (1885),  S.  91.  Biol.  Centralb.  Bd.  v.  (1885-86),  S.  316.  See 
also  Voit,  Ibid.  Bd.  vi.  (1886-87),  S.  243. 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.        123 

Glycerin  (Glycerol).     C3H5(OH)3. 

As  already  stated,  glycerin  is  a  triatomic  alcohol,  the  neutral  fats 
being  ethereal  salts  formed  from  it  with  the  radicles  of  the  higher 
fatty  acids  and  oleic  acid. 

When  pure,  glycerin  is  a  viscid,  colourless  liquid,  of  a  well- 
known  sweet  taste.  It  is  soluble  in  water  and  in  alcohol  in  all 
proportions,  insoluble  in  ether.  Exposed  to  very  low  temperatures 
it  becomes  almost  solid ;  it  boils  at  290°  and  may  be  distilled 
without  decomposition  in  the  absence  of  air. 

It  dissolves  the  alkalis  and  alkaline  earths,  also  many  oxides, 
such  as  those  of  lead  and  copper ;  many  of  the  fatty  acids  are 
also  soluble  in  glycerin. 

It  possesses  no  rotatory  power  on  polarised  light. 

It  is  easily  recognised  by  its  ready  solubility  in  both  water  and 
alcohol,  its  insolubility  in  ether,  its  sweet  taste,  and  its  reaction 
with  bases.  When  sufficiently  heated,  especially  in  presence  of  a 
dehydrating  agent,  glycerin  is  decomposed,  loses  two  molecules  of 
water  and  yields  acrolein.  C3H5(OH)3  =  C3H40-f  2H2O.  This 
substance  possesses  an  intensely  penetrating,  irritating  and  pungent 
odour  so  that  its  formation  enables  glycerin  to  be  readily  identi- 
fied. It  is  the  cause  of  the  peculiar  smell  arising  from  overheated 
fats.  Chemically  it  is  the  aldehyde  of  allyl  alcohol  (derived  from 
the  defines)  and  is  intermediate  between  this  substance  and  acry- 
lic acid,  which  is  a  homologue  of  oleic  acid.     (See  above.) 

Glycerin  is  formed  in  traces  during  the  alcoholic  fermentation 
of  sugar  1.  It  is  prepared  in  bulk  by  distilling  in  a  current  of 
superheated  steam  the  fluid  residue  left  after  the  saponification  of 
fats  with  lime. 

Soaps. 

When  neutral  fats  are  heated  with  lime  or  caustic  alkalis  under 
pressure  they  are  decomposed,  the  metal  combining  with  the  free 
fatty  or  oleic  acid  to  form  a  salt,  leaving  the  glycerin  in  solution. 
These  salts  are  called  soaps,  being  soluble  in  water  if  the  metal  is 
an  alkali,  insoluble  if  it  is  calcium,  lead,  or  other  similar  metal. 
The  reaction  which  takes  place  during  the  above  saponification  is 
as  follows. 

Tri-sterin.  Potassium  sterate.  Glvcerin. 

CsHsCCxvHas .  C0.0)3  +  3KH0  =  3(Cx,H35.COOK)  +  C3H5(OH)3. 

A  similar  decomposition  into  glycerin  and  free  fatty  acid  can  be 
effected  by  pancreatic  juice  (see  p.  64),  the  acid  uniting  with  the 
alkali  of  the  juice  or  of  the  bile  to  form  a  soap.  This  decomposi- 
tion is  however  quantitatively  inconsiderable  but  qualitatively  of 
great  importance  for  the  absorption  of  fats,  owing  to  the  extraor- 

1  Pasteur,  Ann.  d.  Chem.  u.  Pharm.  Bel.  cvi.  (1858),  S.  338. 


124 


lIctic  acids. 


dinarily  great  emulsifying  power  of  a  mixture  of  bile,  free  fatty 
acids  and  soluble  soaps.  The  same  decomposition  takes  place 
when  fats,  more  especially  butter,  turn  rancid. 

III.     Acids  of  the  Glycolic  and  Oxalic  Series. 

"When  one  atom  of  hydrogen  in  a  paraffin  is  replaced  by 
hydroxyl  a  primary  monatomic  alcohol  is  obtained ;  if  a  second 
atom  is  replaced  a  parallel  series  of  diatomic  alcohols  may  be  pre- 
pared, which  are  known  as  glycols.  The  replacement  of  a  third 
atom  of  hydrogen  by  hydroxyl  yields  the  triatomic  alcohols  (e.  g. 
glycerin).  Further,  just  as  the  monatomic  alcohols  yield  acids 
by  oxidation,  so  also  do  the  glycols  ;  but  from  the  latter  two  series 
of  acids  can  be  obtained,  known  respectively  as  the  glycolic  and 
oxalic  (succinic)  series.     Thus  at  first : 

Ethyl-glycol.  Glycolic  acid. 

GJI,(OB.),  +  O2  =  CHo(OH) .  COOH.+  H^O. 

By  further  oxidation  a  member  of  the  glycolic  series  can  be 
converted  into  a  member  of  the  oxalic  series,  thus : 

Glycolic  acid.  Oxalic  acid. 

CH^COH)  .  COOH  +  O2  =  (GOOH)^  +  H2O. 

The  acids  of  the  glycolic  series  are  monobasic,  those  of  the 
oxalic  dibasic. 

The  following  table  exhibits  the  above  relationships  in  a  con- 
venient form. 


Paraffin 

Alcohol 

Acid 

Glycol 

Acid  I 

Acid  II 

Methane 

Methyl 

Eormic 

Carbonic  ^ 

CH4 

CHgCOH) 

H .  COOH 

„ 

CO(OH).(OH) 

Ethane 

Ethyl 

Acetic 

Ethyl-Glycol 

Glvcolic 

Oxalic 

C2He 

CaHsCOH) 

CH3.COOH 

C2H4(OH)2 

CH2(0H).C00H 

(C00H)2 

Propane 

Propyl 

Propionic 

Propyl-glycol 

Lactic 

Malonic 

CgHg 

C3H,(0H) 

C2H5  .  COOH 

CgHeiOH), 

CaHifOH) .  COOH 

CH2(COOH)2 

Butane 

Butyl 

Butyric 

Butyl-glycol 

Oxybutyric 

Succinic 

C4H10 

C4H9(OH) 

C3H7 .  COOH 

C4H8(OH)2 

CgHeiOH) .  COOH 

C2H4(COOH)2 

Glycolic  Acid  Seeies. 
Lactic  (hydroxy-propionic)  acid.    CgHeOg. 

This,  after  carbonic  acid,  is  to  the  physiologist  the  most  important 
acid  of  the  series. 

If  lactic  acid  is  regarded  as  derived  from  propionic  acid 
CH3 .  CH2 .  COOH,  it  may  be  noticed  at  once  that  two  isomeric 

1  This  acid  is  frequently  classed  in  the  preceding  group  of  acids  as  the  first  of 
the  glycolic  series. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        125 

lactic  acids  must  be  capable  of  being  formed  from  it.  These  acids 
will  have  the  following  formulae  respectively:  CH3.CH(0H). 
COOH  and  CHg  (OH) .  CH^ .  COOH.  Of  these  the  first  is  known 
as  ethylidene-lactic  acid,  the  second  as  hydracrylic  acid. 

In  addition  to  the  above  a  third  acid,  isomeric  with  ethylidene- 
lactic  acid  is  known,  namely  sarcolactic  or  paralactic  acid.  Of 
these  three  acids  only  two  occur  in  the  body,  hydracrylic  being 
absent.  A  fourth  acid,  to  which  the  name  of  ethylene-lactic  acid 
has  been  given,  has  also  been  described  as  isomeric  with  hydra- 
crylic acid.  It  is  however  probable  that  this  acid  is  really  acetyl- 
lactic  acid,  hydracrylic  acid  being  the  true  ethylene-lactic  acid. 
(See  below.) 

The  several  forms  of  lactic  acid  are  all  syrupy  colourless  fluids, 
soluble  in  all  proportions  in  water  and  in  alcohol,  and  to  a  slight 
extent  in  ether.  They  possess  an  intensely  sour  taste,  and  a 
strong  acid  reaction.  When  heated  in  solution  they  may  partially 
distil  over  in  the  escaping  vapour,  but  are  usually  decomposed 
during  the  process.  They  form  salts  with  metals,  of  which  those 
with  the  alkalis  are  very  soluble  and  crystallise  with  difficulty. 
The  calcium  and  zinc  salts  are  of  the  greatest  importance,  as  will 
be  seen  later  on,  inasmuch  as  by  their  varying  solubilities  they 
afford  a  means  of  separating  the  several  acids  each  from  the  other. 

1.     Ethylidene-lactic  acid.     CH3 .  CH(OH) .  COOH. 

This  is  the  ordinary  form  of  the  acid,  obtained  characteristically 
as  the  chief  product  of  the  lactic  fermentation  of  sugars  (see  p.  105). 

From  this  source  it  may  be  readily  prepared  by  adding  a  little  old 
cheese  and  sour  milk  to  a  solution  of  cane  sugar  to  which  some  car- 
bonate of  zinc  is  added.  The  whole  is  kept  warmed  to  40°  or  45°  for 
ten  days  or  a  fortnight,  being  vigorously  stirred  at  frequent  intervals. 
The  lactic  acid  is  fixed  as  a  lactate  by  the  zinc  salt  as  fast  as  it  is 
formed,  this  removal  of  free  acid  being  essential  to  the  progress  of  the 
fermentation  which  does  not  take  place  in  an  acid  solution.  The 
crusts  of  zinc-lactate  formed  during  the  above  process  are  purified  by 
recrystallising,  the  acid  is  then  liberated  from  the  compound  by  the 
action  of  sulphuretted  hydrogen,  and  extracted  by  shaking  up  with 
ether,  in  which  it  is  soluble.  By  a  similar  process  lactic  acid  may  be 
readily  obtained  from  lactose. 

Lactic  acid  occurs  in  the  contents  of  the  stomach  and  intestine, 
more  particularly  during  a  diet  rich  in  carbohydrates,  and  may  be 
readily  formed  by  the  digestion  of  gastric  mucous  membrane  with 
solutions  of  dextrose  or  saccharose.^  According  to  Heintz  ^  it  is 
found  also  in  muscles,  and  according  to  Gscheidlen^  in  the 
ganglionic  cells  of  the  grey  substance  of  the  brain, 

1  Maly,  Ann.  d.  Chem.  u.  Pharm.  Bd.  clxxiii.  (1874),  S.  227. 

2  Ann.  d.  Chem.  u.  Pharm.  Bd.  CLVii.  (1871),  S.  314. 

3  Pfliiger's  Archiv,  Bd.  viii.  (1873-74),  S.  171. 


126  LACTIC   ACIDS. 

The  most  important  salts  of  this  acid  are  those  of  zinc  and 
calcium. 

Zinc  lactate.  Zn  (0311503)2  +  3H2O.  Soluble  in  53  parts  of 
water  at  15°  ;  in  6  parts  at  100°.     Almost  insoluble  in  alcohol. 

Calcium  lactate.  OA  (0311503)2  +  SHgO.  Soluble  in  9-5  parts 
of  cold  water ;  soluble  in  all  proportions  in  boiling  water.  In- 
soluble in  cold  alcohol. 

2.     Sarcolactic  acid. 

This  form  of  the  acid  is  isomeric  with  the  preceding  one.  In 
its  general  chemical  behaviour  as  tested  by  the  various  decom- 
positions it  can  undergo  it  is  found  to  be  identical  with  ethylidene- 
lactic  acid,  the  sole  observable  difference  being  in  the  different 
solubility  of  its  calcium  and  zinc  salts.  But  both  sarcolactic  acid 
and  its  salts  differ  strikingly  from  the  preceding  acid  and  its  salts 
as  regards  their  physical  properties,  for  the  former  exert  a  distinct 
rotatory  action  on  polarised  light  while  the  latter  do  not.  This 
peculiar  kind  of  isomerism,  chemical  identity  with  physical 
difference,  has  been  called  '  physical  isomerism '  to  distinguish  it 
from  the  ordinary  form  of  chemical  isomerism.  It  is  now  more 
usually  and  correctly  called  '  stereochemical  isomerism '  in  accord- 
ance with  the  theory  which  is  held  as  to  the  nature  and  cause  of 
the  phenomenon.     (See  below.) 

This  acid  has  not  yet  been  prepared  synthetically  and  is  only 
known  as  occurring  characteristically  in  muscles  ^  to  which  it 
gives  their  acid  reaction,^  and  in  blood.^  In  the  latter  it  is  found 
more  particularly,  as  might  be  expected,  after  the  muscles  have 
been  in  a  state  of  contracting  activity.^  It  is  also  found  in  urine, 
very  markedly  in  cases  of  phosphorus  poisoning,  and  in  the  same 
excretion  after  violent  muscular  exertion,^  or  artificial  stimulation 
of  groups  of  muscles,^  and  very  strikingly  after  extirpation  of  the 
liver  in  birds,"  and  frogs.^  It  is  also  stated  to  be  formed  in  vari- 
able and  slight  amount  during  the  lactic  fermentation  of  dextrose.^ 
Lactic  acid  has  also  been  frequently  described  as  a  constituent  of 
various  pathological  fluids ;  in  these  cases  it  is  probable  that  the 
acid  is  often  the  sarcolactic  acid.^*^ 

As  occurring  characteristically  in  muscles  it  is  hence  found  in 

1  Wislicenus,  Ann.  d.  Chem.  u.  Pharm.  Bd.  clxvii.  (1873),  S.  .302. 

2  Liebig,  Ann.  d.  Chem.  u.  Pharm.  Bd.  lxii.  (1847),  S.  326. 

3  Gaglio,  Arch./.  Physiol.  Jahrg.  1886,  S.  400. 

4  Spiro,  Zt.f.  physiol.  Chem.  Bd.  i.  (1877),  S.  111.  Cf.  Vou  Frey,  Arch.  f. 
Physiol.  Jahrg.  1885,  "S.  557.     Also  Marcuse,  loc.  cit.  below. 

5  Colasanti  and  Moscatelli.     See  ref.  in  Maly's  Ber'icht.  1887,  S.  212. 

6  Marcuse,  Pfliiger's  Arch.  Bd.  xxxix.  (1886),  S.  425. 

"<  Minkowski,  Centralh.  f.  d.  med.  Wiss.  1885,  No.  2.  Arch.f.  exp.  Path.  u.  Phar- 
makol.  Bd.  xxi.  (1886),  S.'40. 

8  Marcuse,  loc.  cit.    But  see  Nebelthau,  Zt.  f.  Biol.  Bd.  xxv.  (1889),  S.  123. 

9  Maly,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  1874,'  S.  1567. 

10  Cf.  Maly.     Abst.  in  Maly's  Jahresb.  1871,  S.  333.     Fluid  from  ovarial  cyst. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY, 


127 


large  quantities  in  Liebig's  '  extract  of  meat '  which  is  the  most 
convenient  source  for  its  preparation.^ 

Liebig's  extract  is  dissolved  in  four  parts  of  warm  water.  To  this 
solution  two  volumes  of  90  p.  c.  alcohol  are  added  and  the  precipitate 
is  removed  by  filtration.  The  filtrate,  after  concentration,  is  again 
precipitated  with  four  volumes  of  alcohol.  The  filtrate  from  this 
second  precipitate  is  finally  concentrated,  acidulated  with  sulphuric  acid, 
and  extracted  with  excess  of  ether  which  dissolves  out  the  sarcolactic 
acid.  On  evaporating  off  the  ether  and  dissolving  the  residue  in 
water,  the  pure  acid  may  be  obtained  by  forming  its  zinc  salt,  which 
is  purified  by  crystallisation  and  decomposed  by  sulphuretted  hydrogen. 

For  the  method  of  detecting  and  separating  this  acid  from  urine  see 
Salkowski  and  Leube.^ 

The  zinc  and  calcium  salts  of  sarcolactic  acid  are  much  more 
soluble  both  in  water  and  alcohol  than  are  those  of  ethylidene- 
lactic  acid. 

Zinc  sarcolactate.  Zn  (C3H503)2  +  2H20.  Soluble  in  17-5 
parts  of  water  at  15°  or  964  parts  of  boiling  98  p.  c.  alcohol. 

Calcium  sarcolactate.  Ca  (C3H503)2  _+  4H2O  [  ?  41  HoO].  Solu- 
ble in  12-4  parts  of  cold  water,  soluble  in  all  proportions  in  boiling 
water  or  alcohol. 

The/ree  acid  is  dextro-rotatory,  but  the  true  value  of  {a)^  is 
unknown  owing  to  uncertainty  as  to  the  purity  of  the  acid.  The 
salts  on  the  other  hand  are  all  Isevo-rotatory.  For  the  zinc  salt, 
when  one  part  is  dissolved  in  18  of  water  (a)j)^-7'6°. 


Fig. 2.     Zinc  Sarcolactate.  Fig  3      Calcium  Sarcolactate. 

(After  Kiihne.)  (After  Kuhne.) 

Both  this  acid  and  the  preceding  one  yield  an  intense  yellow 
colouration  when  added  to  an  extremely  dilute  (almost  colourless) 
solution  of  ferric  chloride.     This  reaction  is  sometimes  useful.^ 

1  See  Gamgee,  Physiol.  Chemistry,  Vol.  i.  1880,  p.  361. 

2  Die  Lehre  vom  Ham,  1882,  S.  125. 

3  Uffelmann,  Arch.f.  klin.  Med.  Bd.  xxvi.  (1880),  S.  431. 


128  ,        LACTIC  ACIDS. 

When  the  formula  of  ethylidene-lactic  acid  is  examined  it  is  found 
to  contain  what  is  known  as  an  asymmetric  carbon  atom :    that  is  to 
say,  an  atom  of  carbon  whose  affinities  are  saturated  by  four  dissimilar 
H 

i 
radicles.     Thus  H2C— C— COOH. 

I 
OH 

According  to  the  -hypothesis  of  Van't  Hoff  and  Le  Bel  such  a  sub- 
stance must  be  possessed  of  optically  active  properties,  since  all  sub- 
stances which  do  rotate  the  plane  of  polarised  light  contain  an 
asymmetric  carbon  atom.  It  is  known  however  in  certain  cases,  as  for 
instance  racemic  acid,  that  although  the  substance  contains  one  (or 
more)  asymmetric  carbon  atoms  it  may  still  be  optically  inactive  since 
it  is  composed  of  a  mixture  of  isomeric  bodies  possessing  equal  and 
opposite  rotatory  powers.  From  this  point  of  view  it  is  probable  that 
ethylidene-lactic  acid  may  be  such  a  mixture,  and  that  at  present  only 
one  of  the  optically  active  isomers  of  which  it  is  composed  has  been 
obtained,  viz.  sarcolactic  acid. 

In  support  of  this  view  it  is  interesting  to  notice  that  a  dextro- 
rotatory lactic  acid  can  be  obtained  from  the  optically  inactive 
ethylidene-lactic  acid,  by  applying  to  its  ammonium  salt  Pasteur's 
method  for  the  separation  of  a  mixture  of  isomeric  substances  whose 
rotatory  powers  are  equal  and  opposite.  This  consists  in  growing  the 
organism  Penicillium  glaucum  in  a  dilute  solution  of  the  mixture; 
one  of  the  isomers  is  found  to  be  more  readily  destroyed  by  the  plant 
than  is  the  other,  so  that  at  a  certain  stage  only  one  is  left  in  solution.^ 
When  treated  in  this  way  ethylidene-lactic  acid  yields  a  dextro- 
rotatory solution.^  When  a  current  of  dry  air  is  passed  through  sar- 
colactic (or  ethylidene-lactic)  acid  heated  to  150°,  two  molecules  of  the 
acid  lose  two  molecules  of  water  and  yield  a  solid  crystalline  substance 
known  as  lactide  (C3H402)2.  When  boiled  with  water  this  is  recon 
verted  into  optically  inactive  lactic  acid,  thus  effecting  the  reconver 
sion  of  the  optically  active  into  the  inactive  form  of  the  acid. 

The  Van't  Hof£-Le  Bel  hypothesis  of  what  was  originally  called 
'  physical '  isomerism  is  based  upon  considerations  of  the  spaciat 
relationships  of  the  constituents  of  an  organic  substance;  hence  the 
more  recent  use  of  the  expression  '  stereochemical '  instead  of 
'physical.'^ 

The  acid  reaction  of  dead  muscle  is  undoubtedly  due  to  the 
presence  of  sarcolactic  acid,  as  was  first  clearly  shown  by  Liebig 
in  1847.^  In  certain  cases  the  reaction  of  muscle  which  is  still 
irritable  may  become  acid,  and  this  has  usually  been  regarded  as 
due  to  the  development  of  this  acid  during  its  activity.     In  recent 

1  Compt.  Rend.  T.  li.  (1860),  p.  153, 

2  Lewkowitsch,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  1883,  S.  2720. 

3  See   Miller's    Elements  of  Chem.   (Armstrong   and  Groves),  Part  III.  Sec.    l  , 
(1880),  p.  983,  for  details  of  the  Van't  Hoff-Le  Bel  hypothesis. 

*  That  living  (irritable)  muscle  in  a  state  of  rest  is  reallv  alkaline  was  first 
demonstrated  by  Du  Bois  Reymond  in  1859.  Monatsber.  d.  Berl.  Akad.  1859,  S. 
288.     See  his  Gesammel.  Abhdl.  Bd.  11.  1877,  S.  3. 


CHEMICAL  BASIS   OF   THE   AKIMAL  BODY.        129 

times,  notwithstanding  the  evidence  of  the  production  of  large 
amounts  of  sarcolactic  acid  during  muscular  contraction  (see 
above),  the  view  has  been  put  forward  that  the  acid  reaction  of 
contraction  is  due  rather  to  other  substances,  as  for  instance  acid 
phosphates,  than  to  the  acid.^  This  view  is  by  no  means  proved 
and  is  incompatible  with  the  preponderating  evidence  of  the  re- 
searches already  quoted  on  the  relationships  of  this  acid  to  mus- 
cular activity,  and  of  more  recent  observations.^  It  is  possible 
that  the  acid  reactiui^  of  active  muscle  is  of  complex  origin,  being 
partly  due  to  lactic  acid,  which  by  acting  on  an  alkaline  phos- 
phate may  convert  it  into  an  acid  salt,  while  finally  there  is  an 
excess  of  the  lactic  acid,  most  marked  in  rigor. 

There  is  but  little  doubt  that  the  glycogen  normally  present  in 
muscles  is  diminished  in  amount  during  their  contracting  activity. 
and  it  has  been  frequently  urged  that  the  acid  reaction  of  muscle 
is  due  to  the  formation  of  sarcolactic  acid  from  this  glycogen. 
This  view  seems  to  rest  entirely  on  the  fact  that  during  activity 
glycogen  disappears  and  lactic  acid  is  formed,  but  is  devoid  of 
convincing  experimental  evidence.  It  is  known  that  a  muscle 
free  from  all  glycogen  can  become  acid  during  activity,  and  bear- 
ing in  mind  that  the  acidity  of  active  muscle  is  proportional  to  its 
power  of  doing  work,  and  to  the  work  it  is  called  upon  to  do,'^  it  is 
most  probable  that  the  lactic  acid  is  a  product  of  the  breaking 
down  of  the  complex  (nitrogenous)  molecule  whose  decomposition 
is  the  source  of  the  energy  which  the  muscle  can  set  free.* 
Glycogen  is  according  to  this  view  to  be  regarded  rather  as  a  con- 
venient accessory  to  the  activity  than  as  either  the  basis  of  this 
activity  or  of  the  lactic  acid  which  arises  during  the  activity. 

3.     Ethylene-lactic  acid.    CH2(0H) .  CHo .  COOH. 

This  acid  has  been  usually  described  as  accompanying  sarcolac- 
tic acid  in  extracts  of  muscles,  and  as  being  isolable  from  this  by 
taking  advantage  of  the  varying  solubilities  of  the  zinc  salts  of 
the  two  acids.^ 

More  recent  researches  have  however  made  it  probable  that 
what  has  usually  been  described  as  ethylene-lactic  acid,  obtain- 
able from  muscle-extract,  is  really  acetyl-lactic  acid,  CH3 .  CH 
(C2H3O2)  COOH,  the  true  ethylene-lactic  acid  being  hydracrylic 
acid,  which  does  not  occur  in  the  animal  body.^ 

I  Astaschewskv,  Zt.  f.  physioL  Chem.  Bd.  iv.  (1880),  S.  397.  Weyl  u.  Zeitler, 
TSiUBd.  VI.  (1882),  S.  557.  ^...       ,      .     , 

2  Werther.  Pflu^er's  Arch.  Bd.  xlti.  (1890),  S.  63.  Cf.  AVarren,  Pflugers  Arch. 
Bd.  XXIV.  (1881),  S.  391.  ^   .   ,      ,,    ,   , 

3  Heidenhain,  Merhnnhche  Leistnnr/  Wdrmeentwlck.  u.  Stoffumsatz  bet  der  Muskel- 
thdtigkeit.  Leipzig.  1864.  Eanke,  Te^awMs.  Leipzig,  1865.  Hermann,  C/nto's.  m.  rf. 
Stoffwechsp].  d.  Miiskeln.     Berlin,  1867. 

■*  Cf.  Werther,  loc.  cit,  S.  85.     Halliburton,  .77.  Ph)/siol.  Vol.  viii.  (1887),  p.  154. 
5  Wislicenus,  Ann.  d.  Chem.  n.  Pharm.  Bd.  CLXVii.  (1873),  S.  302. 
«  Siegfried,  Ber.  d.  d.  chem.  Gesell.  Jabrg.  1889,  S.  2711. 

9 


130  OXALIC  ACID. 

Hydroxy-butyric  acid.^     CH3 .  CH  (OH)  .  CH^ .  COOH. 

This  acid  is  the  next  homologue  to  the  lactic  acids  in  the 
glycolic  series.  It  is  frequently  found  in  the  urine  of  acute  dia- 
betes, usually  accompanied  by  aceto-acetic  acid  [CH3 .  CO .  CH2 . 
COOH].  The  pure  acid  is  sirupy  and  Isevo-rotatory.  {a)-a  =  -23'4. 
For  its  separation  from  urine  and  estimation  see  Klilz'''  and 
Stadelmann.2 

Oxalic  Acid  Sekies. 
Oxalic  acid.    (CO  .  OH)^. 

This  acid  does  not  occur  in  the  free  state  in  the  human  body. 
Calcium  oxalate,  however,  is  a  not  unfrequent  constituent  of  urine, 
and  enters  into  the  composition  of  many  urinary  calculi,  the  so- 
called  mulberry  calculus  consisting  almost  entirely  of  it,  and  it  is 
very  commonly  found  in  urinary  deposits.  As  ordinarily  precipi- 
tated from  solutions  of  calcium  salts  by  the  addition  of  a  salt 
of  oxalic  acid,  the  calcium  oxalate  is  usually  amorphous.  To 
obtain  it  in  the  crystalline  form  dilute  solutions  of  the  two 
reagents  must  be  allowed  to  mix  very  slowly,  as  by  diffusion.  In 
urine  the  case  is  different ;  the  oxalate  is  at  first  in  dilute  solution, 
probably  dissolved  by  the  sodium  dihydric  phosphate  (IsraH2P04) 
to  which  the  acidity  is  normally  due.  On  standing  the  urine 
cools  and  the  oxalate  separates  out  in  a  crystalline  form,  viz. 
rectangular  octohedra,  which  is  characteristic  and  striking,  and 
usually  unlike  that  of  any  other  constituent  of  urinary  deposits. 


Pig.  4.     Calcium  Oxalate.     (After  Funke.) 

In  some  cases  it  presents  the  anomalous  forms  of  rounded 
lumps,  dumb-bells,  or  square  columns  with  pyramidal  ends,  but 
these  forms  are  uncommon. 

The  crystals  are  insoluble  in  ammonia  and  acetic  acid,  but 
readily  soluble  in  hydrochloric  or  other  mineral  acid,  also 
slightly   so   in    solutions    of    acid    phosphates    and    urates    of 

^  See  Neubauer  u.  Vogel,  Analyse  d.  Hams,  1890,  S.  110. 

2  Zt.f.  Biol.  Bd.  XXIII.  (1887),  S.  329. 

3  Ibid.  S.  456. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        131 

sodium.  The  above  characteristics  serve  to  identify  this  salt, 
but  in  practice  the  microscopical  appearance  is  usually  of  most 
use. 

Succinic  acid.     COOH .  CH2 .  CH2 .  COOH. 

This  IS  the  third  acid  of  the  oxalic  series,  being  separated  from 
oxalic  acid  by  the  intermediate  malonic  acid,  CH2(COOH)2.  It 
may  occur  in  the  spleen,  the  thymus,  and  thyroid  bodies,  hydro- 
cephalic and  hydrocele  fluids.  It  has  also  been  stated  to  occur 
normally  in  urine,  but  this  is  very  doubtful,^  as  also  is  the  state- 
ment that  it  is  found  in  this  excretion  after  taking  food  rich  in 
asparagin,  e.  g.  asparagus.^  It  is  obtained  as  a  product  of  the 
putrefaction  of  proteids.^ 

Succinic  acid  crystallises  most  usually  in  the  form  of  large 
four-sided  prisms,  occasionally  as  rhombic  tables.  It  is  soluble 
in  about  20  parts  of  cold  water,  much  more  so  in  hot ;  it  is  also 
soluble  in  alcohol,  more  especially  if  hot,  and  is  but  very  slightly 
so  in  ether. 

The  crystals  melt  at  180°  C,  and  boil  at  235°  C,  being  at  the 
same  time  decomposed  into  the  anhydride  and  water.  The 
alkali  salts  of  this  acid  are  soluble  in  water,  insoluble  in  alco- 
hol and  in  ether. 

Preparation.  Apart  from  the  synthetic  methods,  it  may 
readily  be  obtained  by  the  fermentation  of  malic  *  or  tartaric  ^ 
acids,  which  are  closely  related  to  succinic,  the  former  being 
hydroxy-succinic,  COOH.CHo  .CH(OH)  .  COOH,  and  the  latter 
dihydroxy-succinic  acid,  COOH.  CH(OH)  .CH(OH)  .  COOH. 

Some  of  the  amido-derivatives  of  succinic  acid,  viz.  asparagin 
and  aspartic  acid,  are  of  considerable  interest;  they  will  be 
described  later  on. 


Cholesterin.     C26H44O  or  C25H42O.6 

This  substance  is  described  here  rather  for  the  sake  of  conveni-  ■ 
ence  than  from  its  possessing  any  relationship  to  those  which  have 
preceded  it. 

Cholesterin  is  the  only  alcohol  which  occurs  in  the  human 
body  in  the  free  state.  (The  triatomic  alcohol  glycerin  is  al- 
ways found  combined  as  in   the  fats ;    and  cetyl-alcohol  is  ob- 

1  Salkowski,  Pfliiger's  Arch.  Bd,.  iv.  (1871),  S.  94. 

2  V.  Longo,  Zt.  f.  physiol.  Chem.  Bd.  i.  (1877),  S.  213. 

3  Salkowski,  E.  u.  H.,  Ber.  d.  d.  chem.  Gesell.  1880,  S.  189. 

*  Liebig,  Ann.  d.  Chem.  u.  Pharm.  Bd.  lxx.  (1849),  Sn.  104,  363. 

5  Konig,  Ber.  d.  d.  chem.  Gesell.  1882,  S.  172. 

6  Hesse,  Ann.  d.  Chem.  u.  Pharm.  Bd.  cxcii.  (1878),  S.  175.  Schulze  u.  Barbieri, 
Jn.f.  prakt.  Chem.  Bd.  xxv.  (1882),  Sn,  159,  458. 


132 


CHOLESTERIK 


tained  only  from  spermaceti.)  It  is  a  glittering  white  crystalline 
substance,  soapy  to  the  touch,  crystallising  in  fine  needles  from 
its  solution  in  ether,  chloroform,  or  benzol ;  from  its  hot  alcoholic 
solutions  it  is  deposited  on  cooling  in  rhombic  tables ;  this  is  the 
characteristic  form  and  of  great  importance  for  the  identification 
of  cholesterin. 


Fig.  5.    Cholesterin  Crystals.     (After  Fimke.) 


When  dried  it  melts  at  145°,  and  distils  in  closed  vessels  at 
360°.  It  is  quite  insoluble  in  water  and  in  cold  alcohol,  but 
soluble  in  solutions  of  bile  salts. 

Solutions  of  cholesterin  possess  a  left-handed  rotatory  action 
on  polarised  light,  (a)u  =  -3-5  in  ethereal  solution,  =- 37°  in 
chloroforrnic. 

Cholesterin  occurs  in  small  quantities  in  the  blood  and  many 
tissues,  and  is  present  in  abundance  in  the  white  matter  of  the 
cerebro-spinal  axis  and  in  nerves.  It  is  a  constant  constituent  of 
bile,  and  forms  frequently  nearly  the  whole  mass  of  some  gall- 
stones. It  is  found  in  many  pathological  fluids,  hydrocele,  the 
fluid  of  ovarial  cysts,  &c.,  also  in  fseces  and  milk.^  It  also  oc- 
curs in  the  substance  of  the  crystalline  lens,  more  especially  in 
'  cataract.' 

Preparation.  Gall-stones  supply  the  most  convenient  source 
of  cholesterin.  These  are  pounded,  extracted  with  boiling  water 
and  dissolved  in  boiling  alcohol.  The  solution  is  filtered  through 
a  heated  filter,  and  the  cholesterin  separates  out  in  a  fairly  pure 
condition  as  the  filtrate  cools.  It  is  purified  by  resolution  in  boil- 
ing alcohol  to  which  some  caustic  soda  has  been  added  ;  from  this 
it  again  separates  on  cooling,  and  is  finally  washed  witli  cold  al- 
cohol and  water. 

1  Tolmatscheff,  Hoppe-Sevler's  Med.  Chem.  Unfersuch.  Hf.  2  (1867),  S.  272. 
Schmidt-Miilheim,  Pfliiger's  Arch.  Bd.  xxx.  (1883),  S.  384. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        133 

Cholesterin  is  characterised,  apart  from  its  crystalline  form^  by- 
some  striking  reactions  which  may  be  obtained  even  with  micro- 
scopic quantities. 

(i)  When  the  crystals  are  treated  with  concentrated  sulphuric 
acid  they  usually  turn  violet  or  red.  On  the  addition  of  a  little 
iodine  the  play  of  colours  is  very  marked,  the  crystals  being  vari- 
ously coloured,  —  blue,  red,  green,  violet.^ 

(ii)  When  dissolved  in  chloroform,  the  solution  turns  blood- 
red  on  the  addition  of  an  equal  volume  of  concentrated  sulphuric 
acid :  this  turns  to  blue,  green,  and  finally  yellow,  the  change  of 
colour  being  very  rapid  if  the  solution  is  freely  exposed  to  the  air 
in  an  open  dish.  The  sulphuric  acid  under  the  chloroform  exhibits 
a  green  fluorescence.^ 

(iii)  When  evaporated  to  dryness  on  porcelain  with  a  few  drops 
of  concentrated  nitric  acid,  a  yellow  residue  is  obtained,  which 
turns  red  if  treated,  while  still  hot,  with  ammonia. 

Complex  Nitrogenous  Fats  and  their  Derivatives.^ 

Lecithin.     C44H90NPO9. 

Occurs  widely  spread  throughout  the  body.  Blood  (red-cor- 
puscles),* bile,  and  serous  fluids  contain  it  in  small  quantities, 
while  it  is  a  conspicuous  component  of  the  brain,  nerves,  yolk 
of  egg,  semen,  pus,  white  blood-corpuscles,  and  the  electrical 
.organs  of  the  ray.  It  occurs  also  in  yeast  ^  and  other  vegetable 
cells,  and  in  small  amount  in  milk.^ 

The  presence  of  lecithin  in  the  red  blood-corpuscles  may  prove  to 
be  of  no  inconsiderable  importance  in  connection  with  the  possible 
fixation  by  them  of  carbonic  anhydride.''  Setschenow  has  shown  that 
lecithin  acts  like  a  base  towards  carbonic  anhydride,  each  molecule  of 
the  substance  being  able  to  combine  loosely  with  approximately  one 
molecule  of  the  anhydride  (-092  gr.  lecithin  fixes  2-7  cc.  of  CO2) 
at  a  partial  pressure  of  56  mm.*    Further,  it  is  stated  that  red  blood- 

1  See  figures  in  Funke,  Atlas  d.phi/siol.  Chem.  Leipzig,  1858,  Taf.  vi.  Fig,  2,  3. 
This  work  should  be  consulted  for  the  crystalline  forms  of  all  physiologically  im- 
portant substances.  See  also  Ultzmaun  u.  Hoffmann,  Atlas  d.  Harnsed'nnente.  Wien, 
1872. 

2  Cf.  Burchard,  Inaug.  Diss.  Rostock,  1889.  Abst.  in  Ber.  d.  d.  chem.  Gesell. 
Ref.  Bd.  1890,  S.  752. 

3  For  a  fuller  account  of  the  several  substances  comprised  in  this  group  see 
Gamgee,  Physiol.  Chemistrij,  Vol.  i.  (1880),  p.  425  et  seq. 

*  Cf.  Hoppe-Seyler,  Physiol.  Chem.  1877,  S.  402, 

&  Hoppe-Seyler,  Zt.  f.  physiol.  Chem.  Bd.  ii.  (1878),  S.  427  ;  Bd.  iii.  S.  374. 

6  Tolmatscheff,  also'Schmidt-Miilheim,  loc.  cit.  (sub  Cholesterin). 

^  Al.  Schmidt,  Ber.  d.  slicks.  Gesell.  d.  Wiss.  Bd.  xix.  (1867),  S.  30.  Zuntz, 
Centralb.  f.  d.  med.  Wiss.  1867,  S.  529.  Setschenow,  Ibid.  1877,  S.  625;  1879,  S. 
369  ;  Pfliiger's  Arch.  Bd.  viii.  1874,  S.  20.  Fre'de'ricq,  Compt.  Rend.  T.  lxxxiv.  1877, 
p.  661.     Mathieu  et  Urbain,  Ibid.  p.  1305. 

8  Setschenow,  iMe'm.  de  I'Acad.  Imp.  St.  Petersb.  T.  xxvi.  (1879),  No.  13,  p.  19. 


134  LECITHm. 

corpuscles  contain  about  -75  p.c.  of  lecithin,  ^  hence  100  grm.  red  cor- 
puscles might  therefore  hold  in  loose  combination  rather  more  than 
22  cc.  of  carbonic  anhydride.  It  is  of  course  possible  that  the  lecithin 
does  not  exist  in  a  free  state  in  the  unaltered  corpuscles,  and  is  there- 
fore in  living  blood  incapable  of  playing  the  part  above  ascribed  to  it. 
Still  the  possibility  that  it  may  do  so  is  distinctly  worth  some  con- 
sideration, bearing  in  mind  how  scanty  is  our  knowledge  of  the  real 
conditions  which  determine  the  fixation  of  carbonic  anhydride  by  the 
blood. 

When  pure,  it  is  a  colourless,  slightly  crystalline  substance, 
which  can  be  kneaded,  but  often  crumbles  during  the  process. 
It  is  readily  soluble  in  cold,  exceedingly  so  in  hot  alcohol ;  ether 
dissolves  it  freely  though  in  less  quantities,  as  also  do  chloroform, 
fats,  benzol,  carbon,  disulphide,  &c.  It  is  often  obtained  from  its 
alcoholic  solution,  by  evaporation,  in  the  form  of  oily  drops.  It 
sw^ells  up  in  water  and  during  the  action,  as  observed  under  the 
microscope,  extremely  curious  curling  filamentous  processes  can 
be  seen  to  protrude  from  the  edge  of  the  solid.  These  are  the  so- 
called  '  myelin  forms.'  ^ 

Preparation.  Usually  from  the  yolk  of  egg,  where  it  occurs  in 
union  with  vitellin.  Its  isolation  is  complicated,  and  the  reader 
is  referred  to  Hoppe-Seyler.^ 

Lecithin  is  easily  decomposed ;  not  only  does  this  decomposi- 
tion set  in  at  70°  C,  but  the  solutions,  if  merely  allowed  to  stand 
at  the  ordinary  temperature,  acquire  an  acid  reaction,  the  sub- 
stance being  decomposed.  Acids  and  alkalis,  of  course,  effect  this 
much  more  rapidly.  If  heated  with  baryta  water  it  is  completely 
decomposed,  the  products  being  cholin,  glycerinphosphoric  acid, 
and  barium  stearate.     This  may  be  thus  represented  :  — 

Glycerinphosphoric 
Lecithin.  Stearic  acid.  acid.  Cholin. 

C,,H9oNP09  +  3H20  =  2Ci8H3g02       +      CsHgPOe     +     C5H15NO.,. 

When  treated  in  an  ethereal  solution  with  dilute  sulphuric  acid, 
it  is  merely  split  up  into  cholin  and  distearyl-glycerinphosphoric 
acid.  Hence  it  has  frequently  been  regarded  as  a  sort  of  salt  of 
cholin  with  distearyl-glycerinphosphoric  acid.  It  appears  how- 
ever more  probable  from  the  most  recent  researches  that  ,it  is 
really  an  ethereal  compound  of  this  acid  with  the  cholin.^  It 
appears  also  that  there  probably  exist  other  analogous  compounds 
in  which  the  radicles  of  oleic  and  palmitic  acids  take  part. 

1  Hohlbeck,  Kef.  in  Hoppe-Seyler,  Physiol.  Chem.  1877,  S.  402. 
■^  See  M'Kendrick,  General  Physiologii,  1888,  p.  19. 
^  Hdbch.  d.  phi/s.-path.  chem.  Anal.,  1883,  S.  166. 

*  Hundeshageu,  Jn.  f.  prakt.  Chem.  Bd.  xxviii.  (1883),  S.  219.  Gilson,  Zt.  f. 
physiol.  Chem.  Bd.  xii.  (1888),  S.  58.5. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.         135 

In  accordance  with  these  views  the  constitution  of  lecithin  may 
be  most  adequately  represented  by  the  following  formula :  — 


C3H5 


/>(C„H2„_i02)2 


^O.PO^^^ 

^O.C^H,.  (CH3)3N.OH, 


where  C„H2„.i02  represents  the  radicle  of  a  fatty  acid  which  in 
ordinary  lecithin  appears  to  be  that  of  stearic,  viz.  C18H35O . 


Glycerinphosphoric  acid.    C3H9P06.[C3H5.(OH)2.0.PO(OH)2]. 

Occurs  as  a  product  of  the  decomposition  of  lecithin,  and  hence 
is  frequently  found  in  those  tissues  and  fluids  in  which  the  latter 
is  present.     It  may  occur  occasionally  in  urine.^ 

The  acid  is  dibasic  and  forms  salts  which  are  usually,  so  far  as 
they  are  known,  soluble  in  cold  water,  but  the  lead  salt  is  an  ex- 
ception to  this  rule  and  may  hence  be  used  as  a  precipitant.  The 
salts  are  insoluble  in  alcohol. 

It  may  be  prepared  by  the  decomposition  of  lecithin  when 
boiled  with  caustic  alkalis  or  baryta.  It  may  also  be  synthetised 
by  the  direct  action  of  phosphoric  anhydride  or  glacial  phosphoric 
acid  on  glycerin.  The  formation  by  this  method  may  be  regarded 
as  resulting  from  the  union  of  one  molecule  of  glycerin  with  one 
of  phosphoric  acid  and  elimination  of  one  molecule  of  water. 

r  /OH  -| 

Cholin.  C5H15NO2.    (CH3)3  =  N^^^  .  CHo(OH)  L trimethyloxy- 

ethyl-ammonium  hydroxide. 

Discovered  by  Strecker  ^  among  the  products  of  the  decomposi- 
tion of  pigs'-bile  and  subsequently  of  ox-bile,  whence  the  name 
cholin.  It  does  not  occur  in  the  free  state  except  as  a  product  of 
the  decomposition  of  lecithin,  but  has  been  recently  obtained  in 
extracts  of  the  suprarenals.^  It  is  a  colourless  fluid,  of  oily  con- 
sistence, possesses  a  strong  alkaline  reaction,  and  forms  with  acids 
very  deliquescent  salts.  The  salts  with  hydrochloric  acid  and 
with  the  chlorides  of  platinum  and  of  gold  are  the  most  important. 

Cholin  is  a  most  unstable  body,  mere  heating  of  its  aqueous 
solution  sufficing  to  split  it  up  into  glycol,  trimethylamin  and 
ethylene  oxide. 

Since  it  is  a  product  of  the  decomposition  of  lecithin  it  is  best 

1  Sotnitschewsky,  Zt.  f.  physiol.  Chem.  Bd.  iv.  (1880),  S.  214.  But  see  also 
Eobin,  Arch,  de  Pharm.  T.  ii.  p.  532,  and  Chem.  Centralb.  1888,  S.  186. 

'-2  Ann.  d.  Chem.  u.  Pharm.  Bd.  cxxiii.  (1862),  S.  3.5.3  ;  Bd.  cxlviii.  (1868),  S.  76. 
3  Marino-Zuco,  Rend.  d.  R.  accad.  d.  Lincei,  1888,  p.  835. 


136  CHOLIN.    NEUEIN. 

prepared  from  the  yolk  of  egg.^  The  process  is  elaborate  but 
consists  roughly  in  decomposing  the  residue  of  the  yolk,  left  afte;- 
complete  extraction  with  alcohol  and  ether,  by  boiling  it  for  at 
least  an  hour  with  caustic  baryta.  At  the  end  of  this  period  the 
barium  is  precipitated  by  a  stream  of  carbonic  acid,  the  filtrate  is 
concentrated,  extracted  with  absolute  alcohol,  and  from  this  solu- 
tion the  cholin  is  precipitated  as  a  salt  by  the  addition  of 
platinum  chloride.  It  is  finally  separated  from  this  salt  by  means 
of  sulphuretted  hydrogen. 

Wurtz  2  has  obtained  it  synthetically,  first  by  the  action  of  glycol 
CH2 .  OH 

I  chlorhydrin  on  trimethylamine,  and  then  by  that  of  ethylene 

CH2.CI 
oxide  on  a  concentrated  aqueous  solution  of  trimethylamine. 

Cholin  when  pure  is  an  oily  liquid  with  a  strong  alkaline  re- 
action soluble  in  alcohol  or  ether.  It  yields  crystalline  com- 
pounds with  acids  and  some  salts  of  which  the  double  salts  formed 
with  hydrochloric  acid  and  the  chlorides  of  either  gold  or  platinum 
crystallise  readily  and  are  employed  for  the  detection  and  separa- 
tion of  the  base.  The  platinum  salt  is  readily  soluble  in  water, 
insoluble  in  alcohol.  The  gold  salt  is  but  slightly  soluble  in  cold 
water,  but  soluble  in  hot  alcohol. 

When  boiled  in  concentrated  solution  cholin  is  decomposed  into 
glycol  and  trimethylamine. 

(CH3)3  =  ^(^^  =  C,-H,{OIL),  +  N  (CH3)3. 

^CHa .  CH2(0H) 

By  oxidation  with  concentrated  nitric  acid  it  yields  the  ex- 
tremely poisonous  alkaloid  muscarin  CsHisISTOs.^  Cholin  is  itself 
possessed  of  poisonous  properties,  and  arising  as  it  does  from  the 
decomposition  of  lecithin  and  protagon  is  now  recognised  as  one 
of  the  alkaloidal  products  or  ptomaines  (see  below)  which  occur 
in  putrefying  animal  tissues.* 

r                   /OH  -, 

Neurin.     C5H13NO.    (CH3)3  =  ]Sr^^j^ ^jj     ,  trimethylvinyl- 

ammonium  hydroxide. 

This  substance  is  closely  related  to  cholin  both  in  composition 
and  origin,  but  is  much   more  powerfully  toxic  than  that  body. 

1  Diakonow,  for  ref.  and  details  see  Hoppe-Seyler's  Hdbch.  d.  phys.-patli.  chem. 
Anal.  1883,  S.  163. 

2  Ann.  d.  Chem.  u.  Pharm.  Supl.-Bd.  vi.  Sn.  116,  201.  Cf.  Baeyer,  Ibid.  Bd. 
CXL.  (1866),  S.  306. 

3  Schmiedeberg  u.  Harnack,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  vi.  (1876),  S.  101. 
Cf.  Berlinerblau,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  xvii.  (1884),  S.  1139.  But  see 
also  Bohm,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  xix.  (1885),  S.  87. 

*  Brieger,  Zt.'f.  Hin.  Med.  Bd.  x.  (1885),  S.  268.  See  also  Brieger's  works 
referred  to  below,  sub  Ptomaines. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        137 

It  was  first  described  as  a  product  of  the  decomposition  of  pro- 
tagon  by  caustic  baryta,^  and  until  recently  the  names  cholin  and 
neurin  were  applied  interchangeably  to  the  basic  product  of  the 
action  of  baryta  on  lecithin  or  protagon  first  described  under  the 
name  cholin.^  The  researches  of  Brieger  have  however  shown 
that  neurin  differs  distinctly  both  in  composition  and  properties 
from  the  older  cholin,  and  have  further  identified  it  as  one  of  the 
most  commonly  occurring  and  actively  toxic  of  the  alkaloidal  basic 
products  of  the  putrefactive  decomposition  of  animal  tissues  known 
under  the  name  of  the  ptomaines^  (see  below).  Like  cholin  it 
is  in  the  pure  state  a  sirupy  fluid,  with  strongly  alkaline  reaction 
and  is  extremely  soluble  in  water.  It  forms  with  hydrochloric 
acid  and  platinum  chloride  characteristic  double  salts  which  crys- 
tallise readily.  The  double  salt  which  neurin  forms  with  gold 
chloride  crystallises  in  yellow  needles ;  it  is  but  slightly  soluble 
in  cold  water,  though  soluble  in  hot  water 

Protagon.     C160H308N5PO35  ( ?)• 

A  crystalline  substance,  containing  nitrogen  and  phosphorus, 
obtained  by  Liebreich*  from  the  brain  and  regarded  by  him  as  its 
principal  constituent.  The  researches  of  Hoppe-Seyler  and  Diak- 
onow  tended  to  show  that  protagon  was  merely  a  mixture  of  leci- 
thin and  cerebrin.  A  repetition  of  Liebreich's  experiments  has 
however  led  Gamgee  and  Blankenhorn  ^  to  confirm  the  truth  of 
his  results,  and  further  confirmation  has  been  afforded  still  more 
recently.^  Protagon  appears  to  separate  out  from  warm  alcohol 
on  gradual  cooling  in  the  form  of  very  small  needles,  often  arranged 
in  groups  :  it  is  slightly  soluble  in  cold,  more  soluble  in  hot  alcohol, 
and  in  ether.  It  is  insoluble  in  water,  but  swells  up  and  forms  a 
gelatinous  mass.     It  melts  at  200°  and  forms  a  brown  sirupy  fluid. 

Preparation.  Finely  divided  brain  substance,  freed  from  blood- 
vessels and  connective  tissue,  is  digested  at  45°  C.  with  alcohol 
(85  p.  c.)  as  long  as  the  alcohol  extracts  anything  from  it.  The 
united  extracts  are  filtered  while  hot,  and  the  protagon  separates 
out  from  the  filtrate  on  cooling  to  0°.  It  is  next  thoroughly  ex- 
tracted with  ether  to  get  rid  of  all  cholesterin  and  other  bodies 
soluble  in  ether,  and  finally  purified  by  repeated  crystallisation 
from  warm  alcohol. 

By  treatment  with  boiling  solution  of  caustic  baryta  protagon  is 

1  Liebreich,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  ii.  (1869),  S.  12. 

-  No  distinction  is  made  between  cholin  and  neurin  in  the  latest  edition  (1883) 
of  Hoppe-Seyler's  Handbuch  d.  pfu/s.-path.  chem.  Anal. 

3  Brieger,  Ber.  d.  d.  chem.  Gesell.  Jahrg.  xvi.  (1883),  Sn.  1190,  1406;  xvii.  Sn. 
516,  1137. 

■*  Ann.  d.  Chem.  u.  Pharm.  Bd.  cxxxiv.  (1865),  S.  29. 

5  Jl.  of  Physiol.  Vol.  ii.  (1879),  p.  113.  Also  in  Zt.  f.  physiol.  Chem.  Bd.  in. 
(1879),  S.  260.     Gives  history  and  literature  of  the  subject  to  date. 

6  Baumstark,  Zt.f.  physiol.  Chem.  Bd.  ix.  (1885),  S.  145. 


138  CEEEBEIK      CHAECOT'S   CKYSTALS. 

decomposed,  yielding  the  several  products  which  result  from  the 
decomposition  of  lecithin  under  the  same  conditions,  together  with 
an  additional  product  known  as  cerebrin. 

Cerebrin.^    Ci.'RssNOsi?). 

Is  found  in  nerves,  in  pus  corpuscles,  and  largely  in  the  brain. 
In  former  times  many  names  were  given  to  the  substance  when  in 
an  impure  state,  ex.gr.  cerebric  acid,  cerebrote,  &c.  It  was  first 
prepared  by  W.  Miiller  ^  who  constructed  the  above  formula  from 
his  analysis;  the  mean  of  these  is  C,  68-45.  H,  11-2.  N,  4-5. 
O,  15*85.  Great  doubts  are  however  thrown  upon  the  purity  of 
Mliller's  preparations  by  the  researches  of  later  observers.  From 
a  later  investigation  it  appears  to  contain  less  nitrogen  than  is 
stated  above,  the  carbon  and  hydrogen  being  the  same  (C,  68  •74. 
H,  10-91.     N,  1-44.     0,  18-91).3 

It  is  prepared  from  brain  substance  by  extraction  with  alcohol 
and  purified  by  recrystallisation  from  this  solvent ;  its  complete 
separation  however  from  lecithin  &c.  is  difficult,  but  is  attained 
by  treating  the  mixture  with  boiling  barium  hydrate  :  this,  while 
it  has  no  effect  on  the  cerebrin,  decomposes  the  lecithin. 

It  is  a  light,  colourless,  exceedingly  hygroscopic  powder,  which 
swells  up  strongly  in  water,  slowly  in  the  cold,  rapidly  on  heating. 
When  heated  to  80°  it  turns  brown,  and  at  a  somewhat  higher 
temperature  melts,  bubbles  up,  and  finally  burns  away.  It  is  in- 
soluble in  cold  alcohol,  or  ether ;  warm  alcohol  dissolves  it  readily. 
Heated  with  dilute  mineral  acids,  cerebrin  yields  a  sugar  which 
has  recently  been  shown  to  be  identical  with  galactose.  (See 
above  p.  106.) 

Charcot's  Crystals. 

These  remarkable  crystals,  whose  chemical  nature  and  signifi- 
cance have  been  the  subject  of  much  surmise,  were  first  described 
by  Charcot*  in  the  spleen  and  blood  of  leukhaemic  patients. 
Later  researches  have  confirmed  their  characteristic  appearance  in 
this  disease,  and  have  further  shown  that  they  occur  in  health, 
more  particularly  in  semen,  but  also  in  various  tissues ;  ^  they  are 
also  found  in  asthmatic  expectorations.  They  may  be  readily 
obtained  from  semen  by  extracting  with  warm  water,  to  which  a 
little  ammonia  had  been  added,  the  residue  which  remains  after 

1  See  Gamgee,  Physiol.  Chem.  Vol.  i.  p.  439. 

^  Ann.  d.  Chem.  u.  Pharm.  Bd.  cv.  (1858),  S.  361. 

3  Geoghegan,  Zt.  f.  physiol.  Chem.  Bd.  in.  (1879),  S.  332.  See  also  Parous,  Jn. 
f.  prakt.  Chem.  (N.F.)  Bd.  xxiv.  (1881),  S.  310. 

*  Compt.  Rend.  Soc.  Biol.,  1853.     Gaz.  Held.  1860,  p.  755. 

5  Zenker,  Arch.  f.  klin.  Med.  Bd.  xviii.  (1876),  S.  125.  Schreiner,  Ann.  d. 
Chem.  u.  Pharm.  Bd.  194  (1878),  S.  68.  Cf.  Maly's  Jahresb.  Uber  Thierchemie, 
1878,  S.  86. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BQDY. 


139 


semen  has  been  treated  with  boiling  alcohol.  The  crystals  sepa- 
rate out  from  this  solution  on  concentration,  and  may  be  purified 
by  recrystallisation. 


Tig.  6.     Charcot's  Crystals.     (Krukenberg.) 

The  crystals  are  insoluble  in  alcohol,  ether,  and  chloroform, 
slightly  soluble  in  cold  and  readily  so  in  hot  water.  Dilute  acids 
and  alkalis  also  dissolve  them  readily. 

It  has  been  stated  that  the  crystals  are  in  reality  a  compound 
of  phosphoric  acid  with  a  nitrogenous  base  to  which  the  name 
spermin^  has  been  given,  and  the  formula  C2H5N(?)  has  been 
assigned.  This  base  is  obtained  by  the  addition  to  the  crystals 
of  baryta  water  which  forms  a  phosphate  of  barium  and  liberates 
the  base.  It  is  soluble  in  water  and  alcohol,  yielding  strongly 
alkaline  solutions ;  it  may  be  reconverted  into  Charcot's  crystals 
by  the  action  of  phosphoric  acid.^  This  base  was  at  one  time 
regarded  as  closely  related  to,  if  not  identical  with  ethylinimine 
C2H4 .  NH.2  It  has  however  been  recently  shown  that  the  two 
substances  are  not  identical,  and  it  has  further  been  stated  that 
the  composition  of  spermin  is  most  probably  represented  by  the 
formula  CioH26N"4.^ 


AMIDES  AND   AMIDO-ACIDS.     THEIR  DERIVATIVES 
AND   ALLIES. 
Amido-acids  of  the  Acetic  Series. 
1.     Amido-formic  acid.    NH„ .  COOH. 

This  substance  is  identical  with  carbamic  acid,  one  of  the  amido- 
derivatives  of  carbonic  acid,  the  first  acid  of  the  oxalic  acid  series. 
It  will  be  described  under  the  oxalic  group. 

^  Schreiner,  loc.  cit. 

2  Ladenburg  u.  Abel,  Ber.  d.  d.  chem.  GeselL  Jahrg.  xxi.  (1888),  S.  758.  Ethy- 
linimine appears  (see  next  ref.)  to  be  nothing  but  piperazine,  Hof man's  diethylene- 
diamine. 

3  See  Majert  u.  Schmidt,  Ibid.  Jahrg.  xxiv.  (1891),  S.  241.  Poehl,  Ibid. 
S.  359. 


140 


GLYCTN.     SAEKOSIN. 


2.  G-lycin.  C^HsNOa.  [CH^  (NH^) .  COOHJ.  (Amido-acetic  acid.) 
(^Also  called  GlycocoU  and  Glycocine.) 

Does  not  occur  in  the  free  state  in  the  animal  body,  but  enters 
into  the  composition  of  several  important  substances,  more  espe- 
cially hippuric  and  glycocholic  acids.  It  is  also  a  product  of  the 
action  of  hydriodic  acid  on  uric  acid,  and  of  boiling  acids  and 
caustic  alkalis  on  gelatin :  hence  the  name  glycocoll  or  gelatin- 


FiG,  7.     Glycin  Crystals.     (After  Funke). 

sugar,  since  it  possesses  a  sweet  taste.  It  crystallises  in  large, 
colourless,  hard  rhombohedra,  or  four-sided  prisms,  which  are 
easily  soluble  in  water  (1  in  4-3),  insoluble  in  cold,  slightly  solu- 
ble in  hot  alcohol,  insoluble  in  ether. 

Its  solutions  possess  an  acid  reaction,  but  a  sweet  taste.  Glycin 
has  also  the  characteristic  property  of  uniting  with  both  acids  and 
bases,  to  form  crystallisable  compounds,  as  also  with  salts.  In 
this  it  exhibits  its  amidic  nature,  which  is  further  clearly  evi- 
denced by  the  method  of  its  synthetic  production  by  the  action 
of  monochloracetic  acid  on  ammonia :  — 

CH2  (CI) .  COOH  +  2NH3  =  CHo  (NH2)  •  COOH  +  NH4  CU 

Preparation.  Either  synthetically  as  above  or  more  usually  by 
the  decomposition  of  hippuric  acid  by  prolonged  boiling  with 
hydrochloric  acid,  whereby  it  is  split  up  into  glycin  and  benzoic 
acid,  the  latter  being  separated  by  crystallisation  and  shaking  up 
with  ether  in  which  glycin  is  insoluble. 


CsH.NOa.  [CH2 .  NH  (CH3) .  COOH].    (Methyl- 


3.     Sarkosin. 

glycin.) 

Like  glycin  in  its  general  chemical  properties  it  further  resem- 
bles it  in  that  it  is  never  found  in  the  free  state  as  a  constituent 

1  Mauthner  u.  Suida,  Monatshefle  f.  Chem.  Bd.  xi.  (1890),  S.  373. 


CHEMICAL   BASIS  OF   THE   ANIMAL  BODY.        141 

of  the  animal  body.  It  is  however  a  substance  of  considerable 
interest  and  importance,  not  merely  on  account  of  its  chemical 
relationship  to  kreatin  (see  below)  but  as  having  been  employed 
in  a  well-known  series  of  experiments  intended  to  elucidate  the 
probable  mode  of  formation  of  urea  in  the  body.  It  was  stated 
that  when  sarkosin  is  administered  to  an  animal  in  quantities 
such  that  the  nitrogen  given  as  sarkosin  is  equal  to  the  daily 
output  of  nitrogen  as  urea  by  the  animal,  the  urea  disappears 
from  the  urine  and  is  replaced  by  a  new  substance.^  The  latter 
appeared  to  be  a  compound  of  sarkosin  and  carbamic  acid,  known 
generally  by  the  name  of  methyl-hydantoic  acid,  —  NH2 .  CO  .  N 
(CHg) .  CH2 .  COOH.  This  substance  may  be  regarded  as  arising 
from  the  union  of  one  molecule  of  sarkosin  with  one  of  carbamic 
acid  and  elimination  of  one  molecule  of  water,  or  as  being  urea  in 
which  two  atoms  of  hydrogen  are  replaced  by  methyl  and  a  resi- 
due of  acetic  acid  respectively  :  —  NH2 .  CO .  N  (CH3)  (CHo. COOH). 
The  conclusions  drawn  from  these  observations  were  that  just  as 
methyl-hydantoic  acid  is  supposedly  formed  by  the  union  of 
sarkosin  with  carbamic  acid  and  subsequent  dehydration,  so  also 
would  urea  be  formed  if,  instead  of  sarkosin,  ammonia  were  pres- 
ent, to  unite  with  the  carbamic  acid,  form  ammonium  carbamate 
(NH4 .  ISTHg .  COo)  and  by  loss  of  water  yield  urea.  Subsequent 
repetition  of  these  ingenious  experiments  has  shown  that  they 
are  in  no  way  conclusive,  for  in  most  cases  the  sarkosin  is  largely 
excreted  in  an  unaltered  condition,  methyl-hydantoic  acid  being 
formed  in  very  minute  quantities  if  at  all.^  It  is  further  interest- 
ing to  note  that  the  purely  chemical  reactions  which  most  readily 
yield  methyl-hydantoic  acid  out  of  the  body,  involve  the  inter- 
action of  sarkosin  with  cyanic  compounds  such  as  ammonium  or 
potassium  cyanate.^  Moreover  it  has  been  shown  that  at  the 
temperature  of  the  body  sarkosin  and  urea  in  solution  do  not 
yield  methyl-hydantoic  acid,  although  they  do  in  presence  of 
baryta,  especially  when  boiled.*  These  facts  show  that  Schultzen's 
experiments  do  not  strongly  favour  the  carbamic-acid  origin  of 
urea  ;  they  further  show  that  the  methyl-hydantoic  acid  is  prob- 
ably not  formed  by  a  direct  union  of  sarkosin  and  urea,  and  are, 
from  a  purely  chemical  point  of  view,  rather  in  favour  of  a  cyanic 
origin  of  urea. 

4.     Taurin.    CgH.NSOg.  [CH^  (NH^) .  CH^  (SO, .  OH)  ].    Amido- 
ethylsulphonic  acid. 

Isethionic  acid,  CH2  (OH) .  CH2 .  SO2  (OH),  like  glycolic  acid, 

1  Schultzen,  Ber.  d.  d.  chem.  Gesell.  1872,  S.  578. 

2  Baumann  u.  von  Mering,  Ibid.  1875,  S.  584.     E.  Salkowski,  Ibid.  S.  638. 

Also  Zt.  f.pkysiol.  Chem.  Bd.  iv.  (1880),  Sn.  55,  101.  But  see  also  Schiffer,  Ibid. 
Bd.  V.  (1881 ),  S.  257 ;  Bd.  vii.  (1883),  S.  479. 

3  Baumann  u.  Hoppe-Seyler,  Ber.  d.  d.  chem.  Gesell.   1874,  S.  34.     Salkowski, 
Ibid.  S.  116. 

*  Baumann  u.  Hoppe-Seyler,  loc.  cit.    Baumann,  Z6iW.  S.  237. 


142 


TAURIK 


CH2  (OH).  COOH.  contains  two  hydroxyls  replaceable  by  amidogen 
NH2,  so  that  two  isomeric  amido-derivatives  can  be  formed  from 
it.  Of  these  one  is  amido-isethionic  acid  CH2  (OH) .  CHg .  SO2 
(NH2),  the  other  amido-ethylsulphonic  acid  or  taurin.^ 

Taurin  is  stated  to  occur  in  traces  in  the  juices  of  muscles  and 
of  the  lungs,  but  it  is  known  chiefly  as  a  constituent  of  tauro- 
cholic  acid,  which  is  one  of  the  characteristic  acids  of  bile,  more 
especially  of  the  carnivora,  and  above  all,  of  the  dog. 

It  crystallises  in  colourless,  regular,  four-  or.  more,  usually  six- 
sided  prisms ;  these  are  readily  soluble  in  water,  less  so  in 
alcohol.  The  solutions  are  neutral.  It  is  a  very  stable  com- 
pound, resisting  temperatures  of  less  than  240°  C  ;  it  is  not  acted 
on  by  dilute  alkalis  and  acids,  even  when  boiled  with  them.  It 
is  not  precipitated  by  metallic  salts. 

Preparation.  Ox-bile  is  boiled  for  several  hours  with  dilute 
hydrochloric  acid.  The  fluid  residue  is  separated  from  the  resin- 
ous scum,  and  freed  from  any  remaining  traces  of  bile  acids  by 
means  of  lead  acetate,  the  excess  of  precipitant  being  removed  by 
sulphuretted  hydrogen.  The  final  filtrate  is  then  concentrated  to 
crystallisation,  and  the  taurin  finally  purified  by  recrystallisation 


!FiG.  8.    Taurin  Crystals.     (After  Kuhne.) 

from  water.  The  use  of  the  lead  salt  may  be  omitted  in  many 
cases  and  the  taurin  purified  by  several  crystallisations  from 
water. 

The  behaviour  of  taurin  when  introduced  into  the  alimentary  canal  is 
remarkable.  In  the  case  of  man  the  larger  part  reappears  in  the  urine 
in  combination  with  carbamic  acid  as  tauro-carbamic  acid.  In  dogs 
a  large  part  is  excreted  unaltered,  together  with  some  tauro-carbamic 
acid.     In  herbivora  (rabbit)  on  the  other  hand  a  portion  of  it  is  ex- 

1  Taurin  has  usually  been  regarded  as  identical  with  amido-isethionic  acid. 
This  is  not  the  case.  Seyberth,  Ber.  d.  d.  die  in.  Gesell.  1874,  S.  391.  Erlenmeyer, 
Neu.  Rep.f.  Pharm.  Bd.  xxiii.  (1874),  S.  228. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        143 

creted  in  the  uriue,  but  the  larger  part  is  oxydised,  leading  to  a  large 
increase  of  sulphates  in  the  urine  together  with  some  hyposulphites. 
Injected  suhcutaneously  it  is  largely  excreted  in  an  iinaltered  form.^ 

Tauro-carbamic  acid.  NH2CO  .  NH(CH2)  .  CH2 .  (SOoOH).  The 
remarks  which  have  been  already  made  respecting  the  nature  and  for- 
mation of  sarkosin-carhamic  acid  apply  generally  to  this  acid.  It  is 
most  easily  obtained  as  a  potassium  salt  by  the  action  of  potassium 
cyanate  on  taurin.^ 

5.  Kreatin.  C4H9N3O0.  [NH  :  C^^j^^^^  '  ^^^  •  COOHJ. 
(Methyl-guanidinacetic  acid.) 

By  the  union  of  ammonia  with  cyanamide  a  strongly  alkaline 
base  guanidin  is  obtained  :  CN .  NH2  +  IsTHs  =  NH  .  C(NH2)2  (see 
below).  When  sarkosin  is  employed  instead  of  ammonia  a  similar 
reaction  takes  place,  resulting  in  the  formation  of  kreatin  : 
CN.NH2+CH2.NH(CH3).COOH=NH  :  C(NH2).N(CH3).CH2.COOH.3 
Since  sarkosin  is  methyl-amidoacetic  acid  it  is  at  once  obvious 
that  kreatin  may  be  regarded  as  being  methyl-guanidinacetic 
acid.*  When  cyanamide  is  treated  with  boiling  baryta  water  it 
takes  up  a  molecule  of  water  and  yields  urea,  CN.  (NHo)  +H2O 
r=CO(N'H2)2,  hence  as  might  be  expected,  kreatin  yields  by  simi- 
lar treatment  sarkosin  and  urea.  This  is  to  the  physiologist  the 
most  important  chemical  property  of  kreatin,  bearing  as  it  does  so 
closely  upon  one  possible  source  and  mode  of  formation  of  urea  in 
the  body.     (See  sub  urea.) 

Kreatin  occurs  as  a  constant  and  characteristic  constituent  of 
muscles  and  their  extracts  to  an  amount  which  is  variable,  but 
may  be  taken  as  from  0-2  -  0-3  p.  c.  on  the  weight  of  the  muscle.^ 
It  is  also  found  in  nervous  tissue,  and  is  said  to  occur  in  traces 
in  several  fluids  of  the  body.  It  must  however  be  carefully  borne 
in  mind  that  kreatin  very  readily  loses  a  molecule  of  water  and 
thus  becomes  kreatinin,  and  that  the  latter  with  equal  readiness 
takes  up  a  molecule  of  water  to  form  kreatin.  Hence  the  kreatin 
obtained  during  any  analysis  need  not  at  all  necessarily  imply  its 
presence  as  such  in  the  original  tissue  or  fluid  unless  due  allow- 
ance has  been  made  for  the  possible  effect  of  the  methods  em- 
ployed upon  the  reciprocal  conversions  of  kreatin  and  kreatinin. 
This  is  the  cause  of  the  conflicting  statements  as  to  the  occurrence 
of  kreatin  in  urine ;  as  a  matter  of  fact  this  excretion  always  con- 
tains kreatinin.      It  is   on  the  whole  most   probable  that  any 

1  Salkowski,  Ber.  d.  d.  chem.  Gesell.  1872,  S.  637.  Virchow's  Arch.  Bd.  lviii. 
(1873)  ;   S.  460. 

2  Salkowski,  Virchow's  Arch.  Bd.  lviii.  (1873),  S.  460.  Ber.  d.  d.  chem.  Gesell. 
1873,  Sn.  744,  1191,  1312.     Huppert,  Ibid.  1278. 

3  Volhard,  Sitzh.  d.  bayer.  Akad.  1868,  Hft.  3,  S.  472.  Also  Zt.f.  Chem.  1869, 
S.  318. 

*  Cf.  Horbaczewski,  Wien.  med.  Jahrb.  1885,  S.  459. 
5  Voit,  Zt.f.  Biol.  Bd.  iv.  (1868),  S.  77. 


144 


KEEATIN. 


kreatin  which  may  be  found  in  urine  is  due  to  the  conversion  of 
kreatinin  into  kreatin  during  its  extraction,  since  it  has  been 
shewn  ^  that  the  more  rapidly  the  separation  is  effected,  the  less 


Fig.  9.     Kreatin  Cetstals.     (Krukenberg  after  Kiihne.) 

is  the  quantity  of  kreatin  obtained,  and  the  greater  the  amount 
of  kreatinin. 

In  the  anhydrous  form  kreatin  is  white  and  opaque,  but  crys- 
tallises with  one  molecule  of  water  in  colourless  transparent 
rhombic  prisms. 

The  crystals  are  soluble  in  75  parts  of  cold  water,  extremely 
soluble  in  hot ;  slightly  soluble  in  absolute  alcohol,  they  are  more 
soluble  in  dilute  spirit  and  are  insoluble  in  ether.  The  aqueous 
solutions  are  neutral  in  reaction. 

Kreatin  is  a  very  weak  base,  scarcely  neutralising  the  weakest 
acids,  with  which  it  forms  soluble  crystalline  compounds. 

Preparation.  Most  conveniently  from  '  Liebig's  Extract.'  This 
is  dissolved  in  20  parts  of  water  and  precipitated  by  a  slight  ex- 
cess of  basic  acetate  of  lead.  The  filtrate  is  then  freed  from  the 
lead  salt  by  means  of  sulphuretted  hydrogen  and  concentrated  at 
moderate  temperature  (avoid  boiling)  to  a  thin  syrup.  On  stand- 
ing in  a  cool  place  for  two  or  three  days  the  kreatin  crystallises 
out.  The  crystals  are  removed  by  filtration,  washed  with  88  p.  c. 
alcohol,  and  purified  by  recrystallisation  from  water.^ 

Kreatin  yields  no  very  striking  reactions  by  means  of  which  it 
can  readily  be  identified.  It  reduces  Fehling's  fluid  by  prolonged 
boiling  without  any  separation  of  cuprous  oxide.  On  boiling  in 
presence  of  alkaline  mercuric  oxide,  a  transient  red  colour  is  ob- 
tained and  finally  a  separation  of  metallic  mercury.     The  reac- 


1  Dessaignes,  Jn.  de  Pharm.  et  Chirn.  (3)  T.  xxxii.  (1857),  p.  41. 

■^  The  mother-liquor  from  the  kreatin  may  be  used  for  the  preparation  of 
hypoxanthin  and  sarcolactic  acid.  Drechsel,  Darstell.  phys'ioL-chevi.  Prdparate, 
1889,  S.  29. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        145 

tions  of  kreatinin  on  the  other  hand  are  striking  (see  below),  and 
heiice  kreatin  may  be  identified  with  most  certainty  by  conversion 
into  kreatinin,  and  the  determination  of  the  presence  of  the  latter 
substance.  The  conversion  is  readily  effected  by  boiling  with  di- 
lute mineral  acids,  during  which  process  kreatin  loses  one  molecule 
of  water  :  C4H9N3O2  =  C4H7N3O  +  H.O. 

Mention  has  already  been  made  of  the"  possible  and  very 
probable  genetic  relationship  of  urea  to  muscle-kreatin  (see 
§  484).  This  is  a  question  to  which  brief  reference  will  again 
be  made  under  urea. 

P  /NH CO  -1 

6.     Kreatinin.     C4H7N3O.       NH  :  C  | 

L  \N(CH3).CH2J 

Kreatinin  as  already  stated  is  simply  a  dehydrated  form  of 
kreatin.  It  occurs  normally  as  a  constant  constituent  of  urine, 
varying  however  in  amount  from  0'5  to  4*9  grm.  per  diem  accord- 
ing to  the  amount  of  proteid  food  (meat)  eaten.i  It  is  not  a  nor- 
mal constituent  of  mammalian  muscle  but  is  found  in  the  muscles 
of  some  fishes,^  and  has  been  obtained  from  sweat.^  It  crystal- 
lises in  colourless  prisms  or  tables  according  to  the  conditions 
under  which  the  separation  takes  place  and  the  mode  of  pre- 
paration, and  frequently,  owing  to  imperfect  development,  the 
crystals  assume  a  very  characteristic  '  whetstone '  form. 


Fig.  10.     Kreatinin  Crystals.     (Krukenberg  after  Kiiline.) 

Kreatinin  is  readily  soluble  in  cold  water  (1  in  11  "5),  also  in 
alcohol,  but  is  scarcely  soluble  in  ether.  The  aqueous  solutions 
are  usually  alkaline,  but  some  observers  regard  the  alkalinity  as 
■due  to  impurities.*   It  acts  as  a  powerful  base,  forming  compounds 

^  Voit,  loc.  cit.  (sub  kreatin). 

2  Krukenberg,  Unters.  physiol.  Inst.  Heidelb.     Bd.  ix.  Hf.  1.  (1881),  S.  33. 

3  Capranica,  Bull.  R.  Accad.  med.  Roma,  Ann.  viii.  (1882),  No.  6. 

*  Salkowski,  Zt.f.  physiol.  Chem.  Bde.  iv.  (1880),  S.  133;  xii.  (1888),  S.  211. 

10 


146 


KEEATININ. 


with  acids  and  salts  which  crystallise  well.  Of  these  the  most 
important  is  the  salt  with  chloride  of  zinc  (C4H7N30)2ZnCl2,  both 
on  account  of  its  characteristic  crystalline  form  and  of  its  general 
insolubility  in  comparison  with  the  other  compounds  of  this  sub- 


FiG.  11.     Keeatinin-zinc-chloride  Crystals.     (Krukenberg  after  Kiihne.) 

stance.  Hence  its  formation  is  employed  not  merely  for  the  de- 
termination of  kreatinin  but  for  its  separation  from  solutions.  It 
crystallises  in  warty  lumps  composed  of  aggregated  masses  of 
prisms,  or  fine  needles. 

This  compound  is  formed  when  a  concentrated  neutral  solution 
of  the  zinc  salt  is  added  to  a  not  too  dilute  solution  of  kreatinin, 
and  since  it  is  almost  insoluble  in  alcohol  it  is  frequently  con- 
venient to  employ  alcoholic  rather  than  aqueous  solutions  of  the 
two  substances. 

Preparation}  This  does  not  admit  of  any  useful  brief  descrip- 
tion, but  the  principles  involved  are  the  following :  — 

(i)    By  the  action  of  dilute  boiling  mineral  acids  on  kreatin. 

(ii)  By  concentrating  large  volumes  of  urine  to  a  small  bulk. 
From  this  the  kreatinin  is  obtained  as  a  compound  either  by  the 
addition  of  chloride  of  zinc  or  by  precipitation  with  mercuric 
chloride.  From  these  compounds  it  is  then  separated  by  boil- 
ing with  hydrated  oxide  of  lead,  and  is  finally  purified  by 
crystallisation. 

It  may  also  be  precipitated  by  phospho-tungstic  and  phospho- 
molybdic  acids.^ 

Apart  from  the  characteristic  formation  of  the  compound  with 
zinc  chloride,  kreatinin  yields  several  well-marked  reactions,  of 
which  the  following  are  the  more  striking. 

1  For  details  see  Hoppe-Seyler,  Phys.-path.  chem.  Anal.  1883,  S.  182,  and 
Neubauer  u.   Vogel,  Ham-analyse,  1890,  S.  228. 

^  For  receut  synthesis  see  Horbaczewski,  loc.  cit.  (sub  kreatin). 


CHEMICAL  BASIS   OF  THE   ANIMAL   BODY.        147 

1.  WeyVs  reaction.'^  To  the  suspected  solution  a  few 
drops  of  very  dilute  sodium  nitro-prusside  []Sra2(NO)reCy5]  are 
added,  and  then,  drop  by  drop,  some  dilute  caustic  soda.  If 
kreatinin  is  present  a  fine  but  transient  ruby-red  colour  is  ob- 
tained which  speedily  passes  into  yellow.  If  the  solution  is  now 
acidulated  with  acetic  acid  and  warmed 'it  turns  at  first  greenish 
and  finally  blue.^  This  last  colour  is  due  to  the  formation  of 
Prussian-blue.^  Weyl's  reaction  is  extremely  delicate  and  suf- 
fices to  detect  -0287  p.  c.  of  kreatinin  in  pure  solution,  or  -066  p.  c. 
in  urine.  According  to  Krukenberg  the  reaction  is  best  obtained 
by  adding  the  caustic  soda  first  and  then  a  few  drops  of  concen- 
trated solution  of  the  nitro-prusside.  Guareschi  recommends  the 
use  of  10  p.  c.  solutions  of  the  respective  reagents.* 

When  applied  to  urine  the  absence  of  acetone  should  be  ascer- 
tained, since  it  also  gives  a  similar  ruby-red  colour,  but  no  sub- 
sequent blue  can  be  obtained  from  it,  and  the  solution  when 
yellow  turns  red  again  on  the  addition  of  strong  acetic  acid. 
Hydantoin  or  methyl-hydantoin  also  yields  the  red  colouration. 

2.  Jaffe's  reaction.^  On  the  addition  of  an  aqueous  solution 
of  picric  acid  and  a  few  drops  of  dilute  caustic  soda  an  intense 
red  colouration  is  produced.  This  suffices  to  detect  -1  part  of 
kreatinin  in  5000  of  water.  Acetone  alone  gives  a  similar 
colouration,  but  to  a  comparatively  very  feeble  extent. 

By  prolonged  boiling  of  kreatinin  with  Fehling's  fluid,  reduc- 
tion takes  place,  but  there  is  no  simultaneous  separation  of  cuprous 
oxide,  and  it  appears  that  kreatinin  may  prevent  the  separation  of 
the  oxide  when  the  reduction  is  due  not  to  itself  but  to  such  a 
substance  as  dextrose.^ 

7.     Leucin.     CeHigNO^ .  [CH3 .  (CH2)3CH(NH2)COOH]. 
(«-Amido-caproic  acid.) 

Is  a  characteristic  product  of  the  decomposition  of  proteids  and 
gelatin  whether  by  the  action  of  boiling  acids,  caustic  alkalis,  or 
putrefactive  influences.  It  occurs  normally  in  variable  amounts 
in  the  pancreas,  spleen,  thymus,  thyroid,  salivary  glands,  liver, 
&c.,  and  also  in  plants,  more  especially  in  those  parts  in  which 
reserve  materials  are  accumulated,  such  as  bulbs,  tubers,  and 
seeds.  It  is  also  typically  formed  during  the  tryptic  (pancreatic) 
digestion  of  proteids  to  an  extent  which  amounts  on  the  average 

1  Ber.  d.  d.  chem.  Gesell.  1878,  S.  2175. 

2  Salkowski,  Zt.f.  phjsiol.  Chem.  Bde.  iv.  (1880),  S.  133  ;  ix.  (1885),  S.  127. 

3  Krukenberg,  Verhand.  d.  phi/s.-nied.  Ges.  Wiirzburg,  Bd.  xviii.  (1884),  S.  5. 
Confirmed  by  Salkowski.  Cf.  Colasanti,  Moleschott's  Unters.  Bd.  xni.  (1888), 
Hf.  6. 

*  Ann.  di  chim.  e  difarm.  Ser.  4  T.  v.  (1887),  p.  195. 

5  Zt.  f.  physiol.  Chem.  Bd.  x.  (1886),  S.  399. 

6  Worm  Muller,  Pfliiger's  Arch.  Bd.  xxvii.  (1882),  S.  59. 


148 


LEUCIK 


to  some  8 — 10  p.  c.  on  the  proteid  digested,  and  is  in  this  case 
always  accompanied  by  tyrosin.  It  may  occur  in  the  urine,  more 
particularly  in  cases  of  acute  yellow  atrophy  of  the  liver ;  but  its 
presence  in  this  excretion  in  other  and  more  general  diseased  con- 
ditions of  the  liver  is  by  no  means  so  constant  or  certain  as  it  pre- 
sumably would  be  on  the  common  assumption  that  a  large  part  of 
the  urea  leaving  the  body  is  due  to  its  formation  from  leucin  under 
the  converting  action  of  the  liver.^ 

As  usually  obtained  in  a  more  or  less  impure  form  it  crystal- 
lises in  rounded  fatty-looking  lumps  which  are  often  collected 
together  and  sometimes  exhibit  radiating  striation.  When  pure, 
it  forms  very  thin,  white,  glittering  flat  crystals.  It  is  extremely 
soluble  in  hot  water,  less  so  but  still  very  soluble  in  cold  water, 
soluble  in  alcohol,  insoluble  in  ether.  The  crystals  feel  oily  to  the 
touch,  and  are  without  smell  and  taste.  Leucin  is  particularly 
soluble  in  presence  of  acids  and  alkalis.  The  aqueous  solutions 
are  Isevorotatory,  acid  and  alkaline  solutions  on  the  other  hand 
dextrorotatory. 

Preparation,  (i)  From  horn  shavings  by  prolonged  boiling 
with  sulphuric  acid,  5  of  acid  to   13  of  water.     The  resulting 


-„,^,,/^ ^W 

Fig.  12.    Leucin  Crystals.     (Krukenberg.) 

fluid  is  neutralised  by  baryta  and  filtered,  the  excess  of  baryta 
removed  by  the  cautious  addition  of  dilute  sulphuric  acid,  and 
the  final  filtrate  concentrated  to  crystallisation.  It  is  separated 
from  tyrosin  by  repeated  crystallisation,  taking  advantage  of  the 
great  solubility  of  leucin  and  the  shght  solubility  of  tyrosin. 
(ii)  From  the  products  of  the  tryptic  (pancreatic)  digestion  of 
proteids.  After  prolonged  digestion,  using  thymol  and  salicylic 
acid  to  prevent  putrefaction,  the  fluid  is  filtered,  moderately  con- 
centrated, and  set  aside  to  crystallise  ;  by  this  means  a  large  part 

1  Cf.  Salkowski,  Die  Ldire  vom  Ham,  1882,  S.  427.      Lea.  Jl.  of  Physiol.  Vol. 
XI.  (1890),  p.  258. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.        149 

of  the  accompanying  tyrosin  is  removed.  The  filtrate  is  now 
further  concentrated,  treated  with  excess  of  hot  alcohol,  which 
precipitates  the  peptones,  and  filtered  while  hot.  If  much  leucin 
is  present  a  large  part  of  it  crystallises  out  on  cooling  the  alco- 
holic filtrate,  and  the  rest  on  concentrating  by  slow  evaporation. 
There  is  a  large  loss  of  leucin  by  both  the  above  methods,  and 
the  resulting  product  is  far  from  pure.  To  obtain  pure  leucin 
it  should  be  synthetised  by  the  action  of  ammonia  on  a-brom- 
caproic  acid.^ 

Even  an  approximately  quantitative  separation  of  leucin  from  solu- 
tions where  it  is  mixed  with  other  substances,  e.  g.  an  extract  of  tis- 
sues or  a  digestive  mixture,  is  a  matter  of  great  difficulty.  Advantage 
may  in  some  cases  be  taken  of  its  behaviour  towards  liydrated  oxide  of 
copper,  with  which  it  forms  a  compound.'^ 

For  ordinary  practical  purposes  the  microscopic  appearance  of 
the  crystals  affords  the  most  convenient  means  for  recognising 
leucin,  and  in  this  way  very  minute  traces  may  be  determined 
with  certainty.  The  confirmation  of  the  clue  thus  afforded  by 
the  application  of  chemical  tests  is  however  not  easy  unless  a 
fair  amount  of  material  is  at  hand,  and  that  in  a  pure  condition. 
In  the  latter  case  the  following  tests  may  be  applied,  (i)  When 
carefully  heated  to  170°  leucin  sublimes  and  yields  a  charac- 
teristic odour  of  amylamin.  The  only  other  substance  of  physio- 
logical importance  ordinarily  met  with  which  yields  a  sublimate 
on  heating  is  hippuric  acid,  due  to  its  decomposition  and  the  sub- 
limation of  the  benzoic  acid  thus  set  free,  (ii)  Scherer's  test. 
Only  applicable  to  very  pure  leucin.  The  suspected  substance  is 
evaporated  carefully  to  dryness  with  nitric  acid  on  the  lid  of  a 
platinum  crucible ;  the  residue,  if  it  is  leucin,  will  be  almost 
transparent  and  turn  yellow  or  brown  on  the  addition  of  caustic 
soda.  If  this  be  again  very  carefully  concentrated  with  the  al- 
kali an  oily  drop  is  obtained,  which  runs  over  the  platinum  in  a 
spheroidal  state; 

The  optical  properties  of  leucin  have  not  as  yet  been  fully 
worked  out.  Experiment  shows  that  its  solutions  are  sometimes 
optically  active,  at  other  times  inactive,  dependently  upon  the 
source  and  mode  of  formation  of  the  leucin.  This  corresponds 
to  the  expectations  as  to  its  optical  behaviour  based,  in  ac- 
cordance with  the  Van't  Hoff-Le  Bel  hypothesis,,  upon  its  con- 
stitutional formula.'^ 

The  possible  relationship  of  leucin  to  the  formation  of  urea  in 

1  Hiifner,  J7i.f.  praht.  Chem.  (2)  Bd.  i.  (1870),  S.  6. 

2  Hlasiwetz  u.  Habermann,  Ann.  d.  Chem.  u.  Pharm.  Bd.  clxix.  (1873),  S.  150. 

3  For  details  see  Mauthner,  Zf.  f.  physiol.  Chem.  Bd.  vii.  (1882-83),  S.  222. 
Schulze,  Ibid.  Bd.  ix.  (1885),  S.  100.  Lewkowitsch,  Ber.  d.  d.  chem.  Gesell.  1884, 
S.  1439.  Lippniann,  Ibid.  S.  2835.  Schulze  u.  Bosshard,  Zt.  f.  physiol.  Chem. 
Bd.  x.  (1886),  S.  134. 


150 


CYSTIN. 


the  body  lias  been  already  pointed  out  (\ 
considered  under  urea. 


488).    It  will  be  further 


Amido-acids  of  the  Lactic  Series. 


Cystin.     (CsHeNSOs)^ 
pholactic  acid.^ 


[S .  C(CH3)(NH2)  .  C00H]2.     Amido-sul- 


Is  the  chief  constituent  of  a  rarely  occurring  urinary  calculus 
in  men  and  dogs.  It  may  also  occur  in  renal  concretions,  and  in 
gravel,  and  is  occasionally  found  in  urine,  from  which  it  separates 
out  as  a  greyish  sediment  on  standing.  It  is  prepared  from  this 
sediment,  or  better  still  from  cystic  calculi,  by  solution  in  am- 
monia. This  solution  is  then  allowed  to  evaporate  spontaneously 
and  yields  the  cystin  in  regular,  colourless,  six-sided  tables  of  very 
characteristic  appearance.  Cystin  may  be  separated  from  urine 
by  taking  advantage  of  the  formation  of  a  sodium  salt  of  ben- 
zoyl-cystin  when  it  is  shaken  up  with  a  few  drops  of  benzoyl- 
chloride.2 


Fig.  13.     Cystin  Crystals.     (After  Funke.) 

Cystin  is  insoluble  in  either  water,  alcohol,  or  ether,  readily 
soluble  in  ammonia,  differing  in  this  respect  from  uric  acid,  also 
in  many  alkaline  carbonates  and  in  mineral  acids.     Its  solutions 


1  The  constitution  of  cystin  has  been  variously  stated  by  different  authors,  and 
will  only  be  known  with  certainty  when  its  synthesis  has  been  accomplished. 
Slightly  different  formula  have  been  assigned  to  it,  containing  respectively  5,  6,  and 
7  atoms  of  hydrogen.  The  literature  is  fuUv  quoted  by  Kiilz,  Zt.  f,  Biol.  Bd.  xx. 
(1884),  S.  1.  'Cf.  Baumann,  Zt.  f.  physiol.  Chem.  Bd.  viii.  (1884),  S.'299. 

2  Goldmann  u.  Baumann,  Zt.  f.  physiol.  Chem.  Bd.  xii.  (1888),  S.  254.  Udranskv 
u.  Baumann,  Ibid.  Bd.  xv.  (1891),  S.  87. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        151 

are  strongly  Isevorotatory,  (a)D  =-205*9°   in  hydrochloric  acid 
11-2  p.  c.i  or  if  the  acid  is  dilute  (a)D  =  -214°.2 

Apart  from  the  characteristic  crystalline  form  and  its  solubility 
in  ammonia,  the  fact  that  cystin  is  one  of  the  few  crystalline  sub- 
stances, occurring  physiologically,  which  contain  sulphur,  ren- 
ders its  detection  very  easy.  Thus  when  boiled  with  caustic 
alkalis  a  sulphide  of  the  alkali  is  obtained  which  gives  a  dark 
stain  on  silver  foil ;  also  a  brown  or  black  colouration  appears 
when  cystin  is  boiled  in  a  test-tube  with  a  solution  of  oxide  of 
lead  in  caustic  soda.^ 


Amido-acids  of  the  Oxalic  Seeies. 

1.     Carbamic  acid.    NH2  (COOH). 

Carbonic  acid  is  more  usually  classed  at  the  head  of  the  acids 
of  the  glycolic  (lactic)  series.  It  exhibits  however  a  remarkable 
difference  from  the  remaining  acids  of  this  group,  since  they  are 
all  monobasic,  whereas  carbonic  acid  is  dibasic.  It  may  therefore 
be  more  appropriately  classed  with  the  dibasic  acids  of  the  oxalic 
series.  In  virtue  of  the  two  replaceable  hydroxyls  which  it  con- 
tains, it  yields  two  amido-derivatives,  of  which  the  first  is  car- 
bamic acid,  the  second  urea  (NH2)2  CO  or  carbamide.  Carbamic 
acid  is  a  substance  of  peculiar  interest  to  the  physiologist  on 
account  of  the  important  part  it  is  frequently  supposed  to  play 
in  the  formation  of  urea  in  the  animal  body.  It  is  formed  by  the 
direct  union  of  equal  molecules  of  dry  ammonia  and  carbonic 
anhydride,  a  second  molecule  of  ammonia  uniting  with  it  at  the 
same  time  to  yield  the  ammonium  salt  or  ammonium  carbamate. 
Thus  2NH3  +  CO2  =  NH4  NHoCOo :  simple  dehydration  of  this 
salt  yields  urea  (]S]'H2)2CO.  This  point  will  be  returned  to  further 
on  when  discussing  the  probable  mode  of  formation  of  urea  in  the 
body. 

Carbamic  acid  is  unknown  in  the  free  state ;  its  best  known 
salt  is  that  with  ammonium,  but  many  others  have  been  prepared. 
It  further  appears  that  some  of  its  salts  occur  in  serum,  and  it  is 
also  stated  to  be  formed  during  the  oxidation  of  giycin,  leucin, 
and  ty rosin  by  means  of  potassium  permanganate  in  alkaline 
solution.*     Ammonium  carbamate  is  extremely  soluble  in  water, 

1  Mauthner,  Zt.  f.  physiol.  Chem.  Bd.  vii.  (1883),  S.  225.  Cf.  Drechsel,  Arch. 
f.  Physiol.  Jahrg.  18'91,  S!  247. 

"  Baumann,  loc.  cit.  S.  303. 

^  The  followinj^  literature  may  be  additionallv  consulted  on  the  occurrence  of 
cystin  in  urine.  Zt.  f.  physiol.  Chem.  Bde.  ix.  129!i260;  xii.  254;  xiv.  (1889),  109. 
Virchow's  Arch.  Bd.'  c.  (1885),  S.  416.  Maly's  Jahresb.  1886,  S.  465.  Berl.  klin. 
Wochensrh.  1889,  No.  16.     Zt.f.  klin.  Med.  Bd.  xvi.  (1889),  S.  325. 

*  Drechsel,  Ber.  d.  k.  s.  Gesell.  d.  Wiss.  Leipzig.  Math,  naturiciss.  CI.  Juli.  1875. 
Jn.f.prakt.  Chem.  (2)  Bd.  xii.  (1875),  S.  417;  xvi.  (1877),  S.  180;  xxii.  (1880),  S. 
476.  Arch.  f.  Physiol.  Jahrg.  1880,  S.  550.  But  see  also  Hofmeister,  Pfliiger's 
Arch.  Bd.  xii.   (1876),  S.  337. 


152  ASPAETIC.     GLUTAMIC. 

in  which  solution  it  is  gradually  converted  into  the  carbonate. 
At  ordinary  pressures  when  heated  to  60°  it  is  decomposed  into  am- 
monia and  carbonic  anhydride,  but  under  pressure  at  130°  - 140° 
it  yields  urea.  When  electrolysed  in  cold  aqueous  solution  by  a 
rapidly  and  continuously  commutated  current  the  salt  similarly 
loses  water  and  yields  urea  (Drechsel).  The  dehydration  may  be 
represented  as  taking  place  in  the  following  way :  — 

i.     NH2.CO.O.NH4  +  0  =  NH2.CO.O.NH2  +  H20. 
ii.     NH2 .  CO .  0 .  NH2  +  H2  =  NH2 .  CO .  NH2  +  H2O  ; 

or  by  the  action  first  of  H2  and  then  of  0.^ 

2.  Aspartic  (or  asparaginic)  acid.  C4H7NO4.  [COOH .  CHg .  CH 
(iSTHa)  .  COOH].    Amido-succinic  acid. 

This  acid  is  chiefly  obtained  from  plant  extracts,  and  occurs 
notably  in  beet-sugar  molasses.  It  may  be  synthetised,  but  is 
most  conveniently  prepared  by  boiling  asparagin  with  caustic 
alkalis  or  mineral  acids.  It  is  also  a  typical  product  of  the  action 
of  boiling  mineral  acids  and  caustic  baryta  on  both  vegetable  and 
animal  proteids  (antea  p.  49)  and  of  acids  on  gelatin,^  being 
usually  accompanied  by  its  homologue,  glutamic  acid.  It  is  also 
now  recognised  as  a  product  in  minute  quantities  of  the  pancrea- 
tic digestion  of  fibrin  ^  and  vegetable  glutin,*  although  it  does 
not  occur  as  a  constituent  of  any  animal  tissue  or  secretion.  It 
crystallises  in  rhombic  prisms  which  are  but  sparingly  soluble  in 
cold  water  or  alcohol,  but  readily  soluble  in  boiling  water.  Its 
solutions,  if  strongly  acid,  are  dextrorotatory,  but  if  alkaline,  Isevo- 
rotatory.  It  forms  a  characteristic  readily  cry stalli sable  compound 
with  oxide  of  copper,  which  is  practically  insoluble  in  cold,  but 
soluble  in  boiling  water,  and  may  be  used  for  the  separation  of 
aspartic  acid  from  solutions  in  which  it  is  mixed  with  other 
substances.^ 

3.  Glutamic  (or  glutaminic)  acid.  CsHglSTO^.  (Amido-pyro- 
tartaric  acid). 

This  acid  is  homologous  with  aspartic  acid.  The  circumstances 
and  conditions  under  which  it  occurs  are  in  general  the  same  as 
for  aspartic  acid,  but  it  has  not  as  yet  been  obtained  by  the  action 
of  pancreatic  enzymes  on  proteids  and  is  never  found  in  any 
animal  tissues  or  secretions.  But  as  a  product,  often  to  a  large 
amount,  of  the  artificial  decomposition  of  proteids  it  acquires  some 

1  Cf.  Lud wig's  Festschrift,  1887,  S.  1. 

2  Horbaczewski,  Sitzb.  d.  k.  Akad.  d.  Wiss.  Wien.  Bd.  lxxx.  (2  Abth.)  Juni- 
Hft.  1880. 

3  Eadziejewski  u.  Salkowski,  Ber.  d.  deutsch.  chem.  Gesell.  Jahrg.  vii.  (1874), 
S.  1050. 

*  v.  Knieriem,  Zeitsch.  f.  Biol.  Bd.  xi.  (1875),  S.  198. 

5  Hofmeister,  Sitzb.  d.  L  Akad.  d.  Wiss.  Wien,  Bd.  lxxv.  (1877),  2  Abth. 
Marz-Hft. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.        153 

considerable  importance.     It  is  always  prepared  by  treating  pro- 
teids  with  boiling  mineral  acids.^ 

It  crystallises  in  rhombic  tetrahedra  or  octahedra ;  is  not  very 
soluble  in  cold,  but  readily  soluble  in  hot  water;  insoluble  in 
alcohol  and  in  ether.  Its  aqueous  and  acid  solutions  possess  a 
strong  dextrorotatory  power. 

4.  Asparagin.  C4H8N2O3+  H^O.  [COOH  .  CH^ .  CH  (NH2). 
COiSTHg].     Amido-succinamic  acid. 

Although  asparagin  is  not  found  as  a  constituent  of  the  animal 
body  it  is  a  substance  of  considerable  interest  to  the  physiologist. 
Not  only  is  it  closely  related  to  aspartic  acid,"  into  which  it  may 
be  converted  by  the  action  of  boiling  acids  and  alkalis,  yielding  at 
the  same  time  ammonia,  but  it  undoubtedly  plays  a  most  impor- 
tant part  in  the  constructive  proteid  metabolism  of  plants.  Further 
it  exists  in  not  inconsiderable  amount  in  many  plant-tissues  used 
as  food  by  man,  and  is  known,  like  so  many  of  the  members  of 
the  numerous  class  of  amido-acids  to  which  it  belongs  (leucin, 
glycin,  &c.)  to  give  rise  to  urea  when  taken  into  the  body  of  car- 
nivora,-  and  to  uric  acid  in  that  of  birds.^ 

In  plants  asparagin,  like  leucin,  is  found  chiefly  in  those  parts 
which  afford  a  store  of  reserve  material,  such  as  bulbs,  tubers,  &c.,  and 
the  cotyledons  of  seeds.  The  amount  is  however  largely  increased 
during  germination,  and  it  is  therefore  present  in  frequently  very 
large  quantities  in  seedlings,  as  for  instance  those  of  yellow  lupins 
(30  p.c).  The  increase  in  the  young  growing  plant  is  most  probably 
due  chiefly  to  a  formation  of  asparagin  out  of  the  decomposition  of 
reserve-proteids,  although  some  may  be  formed  synthetically.  The 
amount  is  greatest  when  the  seeds  are  germinated  in  the  dark  and  the 
seedling  subsequently  grown  for  some  time  in  semi-obscurity  and 
shielded  from  the  access  of  carbonic  anhydride.  Under  these  condi- 
tions the  formation  of  non-nitrogenous  (?  carbohydrate)  material  is 
simultaneously  prevented;  and  putting  tlae  two  facts  together  it  ap- 
pears probable  that  the  disappearance  of  asparagin  in  seedlings  grown 
under  ordinary  conditions  is  due  to  its  consumption  for  the  synthetic 
production  of  proteids.*  It  is  conceivable  that  the  amido-acids  and 
amides  may  similarly  play  some  part  in  the  synthetic  metabolism  of 
animal  tissues,  though  to  a  presumably  much  slighter  extent,  bearing 
in  mind  how  in  plants  constructive  metabolism  preponderates  so 
largely  over  the  destructive.^ 

Asparagin  crystallises  readily  in  large  rhombic  prisms  which 
are  not  very  soluble  in  cold,  but  readily  soluble  in  hot  water, 
and  are  insoluble  in  absolute  alcohol  and  in  ether.     Its  solutions 

1  Ritthausen  u.  Kreusler,  Jn.   /.  praU.  Chem.  (2)  Bd.  iii.  (1871),  S.  314. 

2  V.  Knieriem,  Zt.  f.  Biol.  Bd.  x.  (1874),  S.  277. 

3  V.  Knieriem,  Ibid.  xiii.  (1877),  S.  36. 

*  Cf.  Vines,  Ph/siologt/  of  Plants,  pp.  124,  150,  174. 
5  Lea,  Jl.  of  Physiol' Yol.  xi.  (1890),  p.  258. 


154  ASPAEAGIN. 

are  dextrorotatory.  It  may  be  prepared  synthetically,^  but  is 
usually  obtained  by  crystallisation  from  the  expressed  juice  or 
extracts  of  the  seedlings  of  peas,  beans,  or  kipins.^  Mercuric 
nitrate  yields  a  precipitate  with  asparagin  which  may  be  used  for 
its  separation  from  vegetable  extracts.^  Urea-ferment  converts  it 
into  succinic  acid.* 

One  point  of  interest  with  respect  to  asparagin  remains  to  be 
briefly  mentioned.  Seeing  that  in  plants  the  nitrogen  requisite 
for  the  construction  of  proteids  appears  to  be  obtained  largely 
from  asparagin,  is  there  any  evidence  that  in  animals  also  the 
nitrogen  of  this  substance  can  take  the  place  of  that  of  proteids  ? 
The  answer  to  this  question  may  be  stated  as  follows :  When 
asparagin  is  administered  to  carnivora  or  birds  practically  the 
whole  of  it  is  converted  into  urea  or  uric  acid  respectively.^  Thus 
in  carnivora  at  least  there  is  no  diminution  of  proteid  metabolism, 
such  as  is  observed  under  a  gelatin  diet,  when  asparagin  is  added 
to  the  food.  In  herbivora  on  the  other  hand  there  appears  to  be 
somewhat  distinct  evidence  that  a  part  of  the  nitrogen  in  proteids 
may  be  replaced  by  that  of  asparagin.^ 

The  question  as  to  the  importance  of  the  nitrogen  of  asparagin  as  a 
possible  replacer  of  that  of  proteids  arose  first  in  connection  with  the 
dispute  already  referred  to  (p.  122)  on  the  mode  of  formation  of  fats  in 
the  animal  body.  In  the  experiments  of  Weiske  and  Wildt  "^  on  which 
Voit  chiefly  based  his  original  views,  a  diet  of  potatoes  was  largely 
used.  The  amount  of  proteid  in  these  was  calculated  from  the  total 
nitrogen  they  contained,  on  the  assumption  that  there  was  no  nitrogen 
present  in  them  in  any  form  other  than  that  of  proteids.  As  a  matter 
of  fact  potatoes  contain  a  not  inconsiderable  quantity  of  asparagin,^  so 
that  making  allowance  for  this  the  total  amount  of  proteid  given  in 
their  experiments  was  much  less  than  they  supposed,  and  might  not 
have  sufficed  to  account  for  the  fat  stored  up.  This  difficulty  would 
obviously  be  got  over  if  it  could  be  shown  that  the  nitrogen  of  asparagin 
can  play  the  part  of  the  nitrogen  of  proteids.      • 

1  See  recently  Piutti,  Chem.  'Centralh.  Bd.  xtx.  (1888),  S.  1459 
^~  Vma.,  Ann.  de    Chim    et  de  Phijs.   (3)   T.  xxii.   (1847),  p.' 160.      Schulze   u. 
Bosshard,  Zt.  f.  phi/siol.  Chem.  Bd.  ix.  (1885),  S.  420. 
3  Schulze,  JE.,  Ber.  d.  d.  chem.  Gesell.  1882,  S.  2855. 
*  Bufalini,  Ann,  di  chim.  e  di  far  mac.  (4)  T.  x.  (1889),  p.  207. 

5  Von  Knieriem,  loc.  cit.  But  of.  von  Longo,  Zt.  f.  physioL  Chem.  Bd.  i.  (1877), 
o.  213. 

6  Weiske  Z?./.  Biol.  Bd.  xx.  (1884),  S.  277.  Wevl,  Biol.  Centralh.  Bd.  ii. 
(1882-83),  b.  277.  These  give  copious  references  to  literature  up  to  date.  In 
addition  see  Voit,  Sitz.  d.  Bayr.  Akad.  1883,  S.  401.  RGhmann,  Pfliiger's  Arch.  Bd. 
xxxix.  (1886),  S.  21.     (On  storage  of  glycogen.) 

^  Zt.f.  Biol.  Bd.  X.  (1874),  S.  1 

8  Schulze  u.  Barbieri,  Landwirth.  Versuchs-Stat.  Bd.  xxi.  (1877),  S.  63. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        155 

THE   UEEA  AND   UKIC   ACID   GEOUP.i 

1.     Urea.     (NH2)2CO.     {Carhamide). 

This  is  the  chief  nitrogenous  constituent  of  normal  urine  in 
mammalia  and  some  other  animals.  The  urine  of  birds  also  con- 
tains a  small  amount,  more  particularly  on  a  meat  diet.  Average 
normal  human  urine  contains  from  2-5  — 3'2  p.c,  the  average  total 
daily  excretion  varying  from  22 — 35  grams  or  as  a  mean  30  grams. 
It  is  also  found  in  minute  quantities  in  normal  blood  ^  (-025  p.c.) 
serous  fluids,  lymph,  and  aqueous  humour :  it  is  not  usually  met 
with  in  the  tissues  except  that  of  the  liver.^  It  is  never  present 
in  normal  mammalian  muscles,  but  may  make  its  appearance  there 
under  certain  pathological  conditions.  Under  ordinary  conditions 
the  amount  of  urea  in  sweat  is  almost  inappreciable,  but  the  older 
statements  of  its  occurrence  in  this  excretion  have  recently  received 
confirmation,  and  it  appears  that  this  source  of  nitrogenous  loss  to 
the  body  may  have  to  be  taken  into  account.* 


Fig.  14.    Urea  Crystals  separated  by  slow  evaporation  from 

AQUEOUS  SOLUTION.     (After  FuDke.) 

When  pure  it  crystallises  from  a  concentrated  solution  in  the 
form  of  long,  thin  glittering  needles.  If  deposited  slowly  from 
dilute  solutions,  the  form  is  that  of  four-sided  prisms  with  pyrami- 
dal ends ;  these  are  always  anhydrous.  When  the  separation 
occurs  rapidly,  as  for  instance  from  a  strong  alcoholic  solution  on 
a  glass-slide,  the  typical  crystalline  form  is  not  readily  observed, 
but  rather  that  of  irregular  dendritic  crystals. 

Urea  is  very  soluble  in  cold  water,  distinctly  less  soluble  in  cold 
alcohol,  readily  so  in  hot ;  it  is  insoluble  in  anhydrous  ether  and 

1  For  full  details  of  the  reactions,  properties,  and  methods  of  determining  and 
dealing  practically  with  the  members  of  this  group,  consult  in  all  cases  Neubauer  u. 
Vogel,  Analyse  des  Hams.  Salkowski  u.  Leube,  Die  Lehre  vom  Ham.  Hoppe- 
Seyler,  Physiol. -path.  chem.  Analyse. 

■^  Gscheidlen,  Stud,  iiber  d.  Ursprunq  d.  Harnstoffs,  Leipzig,  1871. 

3  But  see  Hoppe-Sevler,  Zt.  f.  physiol.  Chem.  Bd.  v.  (1881),  S.  348. 

4  Argutinsky,  Pfl tiger's  Arch.  Bd,  xlvi.  (1890),  S.  594. 


156 


TJEEA. 


in  petroleum-ether.i     It  possesses  a  somewliat  bitter,  cooling  taste, 
resembling  saltpetre. 

Preparation,  (i)  From  urine  by  concentration  to  a  sirupy 
state,  extraction  of  the  residue  with  absolute  alcohol,  and  concen- 
tration of  the  alcoholic  extract,  by  slow  spontaneous  evaporation 
in  a  warm  place,  until  the  urea  crystallises  out.  This  is  then 
purified  by  recrystallising  from  alcohol,  decolourising  with  char- 
coal if  required.  Or  the  urea  may  be  precipitated  as  nitrate  by 
the  addition  of  pure  colourless  nitric  acid  to  strongly  concentrated 
urine  cooled  to  0°.  The  nitrate  is  then  decomposed  in  water  by 
the  addition  of  barium  carbonate,  and  the  urea  extracted  as  before 
with  alcohol,  (ii)  Synthetically  in  many  ways,  of  which  the 
most  usual  and  convenient  is  by  mixing  equivalent  proportions  of 
ammonium  sulphate  and  potassium  cyanate ;  the  ammonium 
cyanate  thus  formed  is  evaporated  to  dryness,  whereupon  it 
undergoes  a  molecular  transformation  to  urea,  which  is  then  ex- 
tracted with  alcohol :  thus  NH4 .  CON  =  NH., .  CO  .  NH,. . 

It  is  interesting  to  note  that  the  above  synthesis  of  urea,  obtained 
in  1828  by  Wohler,  was  the  first  instance  in  which  a  substance 
ordinarily  elaborated  by  the  specific  activity  of  the  animal  body 
was  artifically  prepared. 

Urea  readily  forms  compounds  with  acids  and  bases  ;  of  these  the 
following  are  important  as  a  means  of  detection  and  identification. 

Nitrate  of  urea.    (^^2)^  CO  .  HNO3. 

Obtained  by  the  addition  of  a  slight  excess  of  pure  colourless 
nitric  acid  to  a  moderately  concentrated  solution  of  urea.  The 
nitrate  should  separate  out  rapidly  in  the  form  of  six-sided  or 
rhombic  tables,  frequently  aggregated  in  piles,  but  the  successful  ■ 
obtaining  of  typical  crystals  requires  some  attention  to  the  con- 
centration of  the  solution. 


Fig.  15.    Crystals  of  Nitrate  of  Urea.     (Krukenberg  after  Kiihne.) 
1  Petroleum-ether  consists  of  the  products,  with  low  boiling-points  (up  to  120"^ 


of  the  distillation  of  ordinary  petroleum, 
name  of  li groin. 


It  is  also  known  commerciallv  under  the 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY. 


157 


The  crystals  are  but  slightly  soluble  in  nitric  acid,  or  alcohol, 
more  soluble  in  cold  water,  and  much  more  so  in  hot  water.  They 
are  insoluble  in  ether. 

Oxalate  of  urea.     [(NH2)2CO]2.H2C204  +  H20. 

Obtained  by  the  addition  of  concentrated  aqueous  solution  of 
oxalic  acid  to  a  concentrated  aqueous  solution  of  urea.     This  salt 


Fig.  16.     Crystals  of  Oxalate  of  Urea.     (Krukeaberg  after  Kiihne.) 

crystallises  out  in  rhombic  tables  closely  resembling  those  of  the 
nitrate,  but  they  are  frequently  aggregated  into  a  characteristic 
prismatic  form.  As  in  the  case  of  the  nitrate  some  care  is  required 
with  respect  to  the  concentration  of  the  respective  solutions  during 
its  preparation. 

The  crystals  are  less  soluble  in  oxalic  acid  than  in  water,  but 
may  in  other  respects  be  taken  as  resembling  those  of  the  nitrate 
in  respect  of  their  solubilities. 

Of  the  many  salts  which  urea  forms  with  other  bases  and  salts 
those  which  it  yields  with  mercuric  oxide  and  nitric  acid  are  of 
most  importance.  When  a  solution  of  mercuric  nitrate  is  added 
to  one  of  urea  a  precipitate  is  formed  which,  dependently  upon  the 
concentration  and  relative  amounts  of  the  two  solutions,  may  con- 
tain some  one  of  three  possible  salts,  consisting  of  [(NH2)2  COJo . 
Hg  (N03)2  united  with  1, 2,  or  3  molecules  of  mercuric  oxide  (HgO). 
When  the  solutions  are  fairly  neutral  and  dilute,  the  salt  with 
3  molecules  of  HgO  is  formed  [(NH2)2  00]^ .  Hg(N03)2 . 3  HgO. 
This  is  the  salt  formed  in  the  reactions  on  which  Liebig's  vol- 
umetric method  for  the  determination  of  urea  is  based. 

The  other  more  wijjortant  reactions  of  Urea. 

1.  Urea  may  be  heated  dry  in  a  tube  to  120°  without  being 
decomposed ;  on  further  raising  the  temperature  it  melts  at  132-6 °i 

1  Reissert,  Ber.  d.  d.  chem.  Gesell.  Bd.  xxiii.  (1890),  S.  2244. 


158  UEEA. 

and  afterwards  gives  off  ammonia,  and  if  heated  to  150°  for  some 
time  is  converted  largely  into  biuret :  2(NH2)2CO=NH2.CO.NH.CO 
(XH2)-hNH3.  On  further  heating  to  a  higher  temperature  (200° ) 
it  is  largely  converted  into  cyanuric  acid.  When  biuret  is  dis- 
solved in  water  and  treated  with  caustic  soda  and  dilute  sulphate 
of  copper  it  yields  the  well-known  pink  colour  employed  for  the 
detection  of  peptones,  and  hence  called  the  'biuret  reaction.'  In 
the  application  of  the  test  to  urea  some  caution  is  requisite  while 
heating  the  suspected  substance  to  avoid  carrying  the  decomposi- 
tion beyond  the  biuret  stage.  When  boiled  in  aqueous  solution 
with  strong  sulphuric  acid  or  alkalis  it  is  gradually  decomposed, 
under  assumption  of  two  molecules  of  water,  into  carbonic  acid  and 
ammonia ;  the  same  decomposition  ensues  by  simple  heating  of  the 
aqueous  solution  in  sealed  tubes,  to  180°.  This  forms  the  basis 
for  the  older  '  Bunsen  method'  of  estimating  urea.  A  similar 
change  (hydration)  is  produced  under  the  influence  of  several 
micro-organisms  which  are  found  in  urine  undergoing  alkaline 
fermentation.  Of  these  the  best  known  is  the  Micrococcus  ureae  ^ 
from  which  a  soluble  hydrolytic  enzyme  may  be  extracted.^  (See 
above,  p.  70.) 

2.  When  treated  with  nitrous  acid,  e.g.  impure  yellow  nitric 
acid,  it  is  decomposed  finally  into  carbonic  anhydride,  nitrogen,  and 
water:  (NH2)2CO -f  2HNO2  =  CO2+2N2 -f  SH^O.  A  similar  de- 
composition is  obtained  by  the  action  of  sodium  hypochlorite  or 
hypobromite :  (NH2)2CO  -f  3NaBrO  =  3NaBr  -f  C02-hN2H-2H20. 
Since  the  volume  of  nitrogen  evolved  is  constant  for  a  given  weight 
of  urea,  this  latter  reaction  forms  the  basis  of  a  method  for  the 
quantitative  determination  of  urea.     (Knop-Hiifner. ) 

3.  When  a  crystal  of  urea  is  treated  with  a  drop  of  concentrated 
freshly  prepared  aqueous  solution  of  furfurol  —  C5H4O2  (aldehyde 
of  pyromucic  acid)  and  then  immediately  with  a  drop  of  hydro- 
chloric acid  (sp.  gr.=  l-10)  a  play  of  colours  is  observed  which 
passes  rapidly  from  yellow  through  green,  blue,  and  violet  to  a  final 
brilliant  purple.  The  test  may  be  also  applied  by  the  addition  of 
three  drops  of  the  acid  to  a  mixture  of  one  drop  of  1  p.c.  aqueous 
urea  solution  and  -5  cc.  of  aqueous  furfurol  solution.^ 

Detection  in  Solutions.  In  addition  to  the  microscopic  appear- 
ance of  the  crystals  obtained  on  evaporation,  the  nitrate  and  oxa- 
late should  be  formed  and  examined.  Another  part  should  give  a 
precipitate  with  mercuric  nitrate,  in  the  absence  of  sodium  chloride 
but  not  in  the  presence  of  this  last  salt  if  in  excess ;  in  presence 
of  sodium  chloride  the  mercuric  nitrate  reacts  first  with  the  sodium 
salt  in  preference  to  the  urea.     A  third  portion  is  treated  with 

1  Pasteur,  Compt.  Rend.  T.  l.  (1860),  p.  869.  Van  Tie^hem,  Ihid.  T.  lviii.  (1864), 
p.  210.     Jaksch,  Zt.   f.  phi/siol.  Chem.  Bd.  v.  (1881),  S.  395. 

2  Musculus,  Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  214.  Lea,  Jl.  of  Phijsiol.  Vol.  vi. 
(1885),  S.  136. 

3  Schiff,  Ber.  d.  d.  chem.  Gesell.  1877,  S.  773. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.         159 

nitric  acid  containing  nitrons  fumes ;  if  urea  is  present,  nitrogen 
and  carbonic  acid  will  be  obtained.  To  a  fourth  part  pure  nitric 
acid  in  excess  and  a  little  mercury  are  added,  and  the  mixture  is 
warmed.  In  presence  of  urea  a  colourless  mixture  of  gases  (N"  and 
CO2)  is  given  off.  A  fifth  portion  is  treated,  after  evaporation  to 
dryness,  in  the  way  above  described  for  the  application  of  the 
biuret  reaction,  and  a  sixth  part  is  tested  with  furfurol. 

Quantitative  determination.  The  methods  are  based  on  some 
of  the  reactions  above  described.  They  consist  of  (i)  Precipita- 
tion by  a  standardised  solution  of  mercuric  nitrate  (Liebig). 
(ii)  Decomposition  into  carbonic  acid  and  nitrogen  by  means  of 
sodium  hypobromite,  and  measurement  of  the  volume  of  nitrogen 
(Knop-Hiifner).  (iii)  Conversion  into  carbonic  acid  and  ammonia 
by  heating  in  a  sealed  tube  with  an  ammoniacal  solution  of  barium 
chloride,  and  determination  of  the  weight  of  barium  carbonate 
obtained.     (Bunsen.) 

Although  simple  in  principle,  the  above  methods,  and  especially 
the  first,  require  the  careful  observance  of  certain  precautions  to 
ensure  accuracy.  The  needful  precautions  have  recently  been 
most  assiduously  investigated,  more  particularly  by  Pfluger  and 
his  pupils,  and  of  these  and  of  the  application  of  the  methods  a 
full  account  is  given  in  Neubauer  and  Vogel's  exhaustive  work  Die 
Analyse  des  Hams. 

The  determination  of  the  total  nitrogen  in  urine  is  also  of  great 
importance,  and  is  now  usually  carried  out  by  Kjeldahl's  method.^ 
This  consists  in  converting  all  the  nitrogen  of  a  measured  portion 
of  urine  into  ammonia  by  boiling  with  fuming  sulphuric  acid  and 
the  subsequent  addition  of  potassium  permanganate.  The  am- 
monia is  then  expelled  from  the  acid  solution  by  distillation  with 
an  excess  of  caustic  soda  or  potash,  the  ammonia  being  received 
into  a  measured  volume  of  standardised  acid,  whose  diminution  of 
acidity  due  to  the  absorption  of  ammonia  is  finally  determined  by 
titration  with  standard  alkali. 


The  synthesis  of  urea  by  molecular  transformation  of  ammonium 
cyanate  indicates  an  undoubtedly  close  relationship  of  urea  to  cyanic 
acid,  and  there  are  other  reactions  which  enforce  the  same  idea. 
Thus  by  the  union  of  water  with  cyanamide,  which  is  readily 
affected  by  treatment  with  50  p.c.  sulphuric  acid,  urea  is  obtained  : 
—  CN .  NH2  +  H2O  =  (NH2)2  CO.  It  is  further  stated  that  when 
potassium  cyanate  and  acid  potassium  tartrate  are  dissolved  in 
water  and  the  mixture  is  kept  for  some  time,  a  not  inconsiderable 
amount  of  urea  is  formed  along  with  some  carbonic  acid,^  thus 
affording  experimental  support  of  Salkowski's  view^  that  urea 

1  Zt.f.  anal.  Chem.  Bd.  xxii.  (1883),  S.  366. 

2  Hoppe-Seyler,  PJujsiol.  Chemie,  S.  809. 

3  Zt.  f.  physiol.  Chem.  Bd.  i.  (1877),  S.  41. 


160  UREA. 

might  arise  in  the  body  from  the  union  of  two  molecules  of  cyanic 
acid  and  one  of  water :  CO.NH+CO.NH+H2O  =  (NH2)2CO-[-C02. 
The  final  formation  of  cyanuric  acid  (C0.IsrH)3  by  the  action  of 
heat  on  dry  urea  is  further  evidence  in  the  same  direction.  On 
the  other  hand  there  are  a  number  of  reactions  resulting  in  the 
production  of  urea,  which  leave  but  little  doubt  that  urea,  while 
closely  related  to  cyanic  acid,  is  truly  the  amide  of  carbonic  or 
carbamic  acid.  Thus  by  the  action  of  ammonia  on  phosgene 
gas  :  —  COOL  +  2NH3  =  CO  (NH2),  4- 2HC1 :  of  ammonia  on 
diethyl-carbonate  :  —  CO.(C2H50)2  +  2NH3  =  C0(NH2)o  +  2C0H5. 
OH  :  —  reactions  which  are  strictly  analogous  to  the  formation  of 
acetamide  CHg .  C0(NH2)  by  the  action  of  ammonia  on  acetyl 
chloride  CH3 .  COCl,  and  on  ethyl-acetate  CH3 .  COO  (C2H5). 

It  is  interesting  to  observe  here  that  acetamide  yields  methylcyanide 
by  treatment  with  phosphorous  pentoxide :  —  CH3 .  CO  (NH,)  ==  CH3. 
CN+H2O. 

Acetamide  is  also  formed  by  the  dry  distillation  of  ammonium 
acetate,  the  change  being  one  of  simple  dehydration ;  and  this  re- 
action is  one  of  general  applicability,  amides  being  formed  by  the 
removal  of  one  molecule  of  water  from  the  ammonium  salt  of  a 
monobasic  acid  or  of  two  molecules  of  water  from  that  of  a  dibasic 
acid,  e.g.  ammonium  oxalate  yields  oxamide.  Now  although  urea 
has  not  been  formed  by  the  dehydration  of  ammonium  carbonate, 
it  is  readily  rehydrated  into  the  carbonate  by  the  action  of  acids, 
alkalis,  superheated  water,  or  the  urea  ferment.  Further,  if  instead 
of  operating  on  ammonium  carbonate  the  ammonium  salt  of  car- 
bamic acid  (see  p.  151)  be  heated  in  sealed  tubes  to  140°,  or  if  it 
be  electrolysed  with  a  rapidly  commutated  current,  it  loses  a  mole- 
cule of  water  and  is  converted  into  urea. 

When  the  purely  chemical  facts  above  stated  are  applied  to  the 
formation  of  urea  in  the  animal  body  it  is  at  once  obvious  that 
urea  might  originate  from  some  cyanic  source,  or  from  a  simple 
dehydration  of  ammonium  carbonate  or  carbamate.  A  full  dis- 
cussion of  the  possibilities  thus  indicated  lies  outside  the  scope  of 
this  work,  but  it  may  not  be  out  of  place  to  indicate,  as  briefly  as 
may  be,  the  various  views  which  have  been  put  forward  concern- 
ing the  probable  way  in  which  urea  originates  in  the  body.^ 

There  is  little  reason  for  doubting  that  the  larger  part  of  the 
nitrogen  which  leaves  the  body  as  urea  was  at  one  time  a  constit- 
uent of  the  nitrogenous  muscle-substance  (see  §  484.)  There  is 
equally  no  doubt,  both  from  general  considerations  and  from  the 
fact  that  no  urea  can  ever  be  detected  in  muscles  normally,  that 
the  nitrogen  does  not  make  its  exit  from  the  muscles  as  ready- 
made  urea.     Neither  until  recently  had  urea  been  obtained  by 

1  The  literature  of  the  subject  is  very  fully  quoted  in  Bunge's  Physiol,  and 
pathol.  Chemistry,  1890.     Lecture  xvi.  pp.  310-348. 


CHEMICAL   BASIS   OF  THE  ANIMAL  BODY.        161 

any  purely  chemical  means  from  the  products  of  the  decomposi- 
tion of  proteids. 

The  older  statements  of  Bechamp  and  Ritter  that  urea  may  be  ob- 
tained from  proteids  by  the  action  of  j)otassium  permanganate  have 
been  shown  to  be  erroneous.^  It  is  at  most  possible  that  a  trace  of 
guanidin  may  be  formed,  and  guanadin  can  by  the  action  of  water  be 
converted  into  urea  and  ammonia:  ]IsrH.C(]SrH2)2-l-H20=(NH2)2CO 
-f-NHs.^  Drechsel  has  however  obtained  from  among  the  products  of 
the  decomposition  of  casein  with  concentrated  boiling  hydrochloric 
acid  and  chloride  of  zinc  a  base  to  which  he  has  given  the  name  of 
'lysatin.'     When  boiled  with  baryta  water  in  excess  it  yields  urea.^ 

What  knowledge  have  we  of  the  possible  or  probable  form  un- 
der which  the  nitrogen  may  make  its  primary  exit  from  the 
muscles  ?  The  connection  of  muscle-kreatin  with  urea-formation 
has  been  already  discussed  (§  484,  485)  and  the  evidence  of  the 
connection  may  be  briefly  summed  up  as  follows.  A  considerable 
amount  of  kreatin  exists  (?)  in  the  muscles  at  any  one  time,  hence 
probably  a  considerable  amount  is  continuously  being  formed ; 
there  is  no  evidence  that  any  of  this  kreatin  leaves  the  body  as 
such,  hence  it  is  presumably  converted  into  some  other  substance 
before  being  discharged,  and  this  other  substance  is  probably  urea, 
seeing  that  kreatin  may  be  readily  decomposed  into  urea  and 
sarkosin.  There  are  further  reasons  for  supposing  that  the  nitro- 
gen leaves  the  muscles  as  a  compound  containing  comparatively 
little  carbon,  and  kreatin  answers  to  this  requirement,  since  it 
contains  only  four  atoms  of  carbon  to  three  of  nitrogen.*  If  this 
latter  view  be  correct  it  implies  that  the  nitrogen  is  not  split  off 
in  the  form  of  amido-acids,  since  there  is  not  sufficient  carbon  in 
proteids  to  convert  their  nitrogen  into  the  amido-acids  with  which 
we  have  to  deal  in  the  body.  On  the  other  hand  when  these 
amido-acids  (glycin,  leucin,  aspartic  acid  and  asparagin)  are  in- 
troduced into  the  body  they  are  partly  converted  into  urea,  so  that 
if  formed  they  would  account  for  a  portion  at  least  of  the  urea 
excreted. 

When  proteids  are  decomposed  by  caustic  alkalis,  more  espe- 
cially baryta,  or  during  putrefaction,  they  yield  much  ammonium 
carbonate,  which  by  simple  dehydration  would  give  urea.  Now 
although  ammonium  carbonate,  like  many  other  salts  of  this  base, 
is  readily  converted  into  urea  when  administered  to  man  or  other 
animals,  there  is  no  evidence,  although  it  is  a  possibility,  that  the 
nitrogen  leaves  the  tissues  as  ammonium  carbonate. 

1  Loew,  Jn.  f.  praht.  Chem.  (2)  Bd.  ii.  (1870),  S.  289.  Tappeiner,  Ko7i.  sacks. 
Gesell.  d.  Wiss.  1871.     See  Abst.  in  Maly's  Bericht.  1871,  S.  11. 

2  Lossen,  Ann.  d.  Chem.  u.  Pharm.  Bd.  cci.  (1880),  S.  369. 

3  Ber.  d.  d.  chem.  Gesell.  1890,  S.  3096.  Cf.  Arch.  f.  Physiol.  Jahrg.  1891,  S. 
254  et  seq. 

*  Bunge,  loc.  cit.  pp.  320,  328. 

11 


162  UEEA. 

The  above  statements  seem  to  embrace  all  that  can  be  suggested 
as  to  the  tissue-antecedents  of  urea,  and  it  remains  now  to  con- 
sider the  probable  mode  and  seat  of  their  conversion  into  urea. 
As  regards  kreatin  it  may  be  that  it  is  split  up  into  urea  and  sar- 
kosin,  the  latter  being,  like  other  amido-acids,  also  converted  into 
urea.  When  the  amido-acids  are  compared  with  urea  it  is  not 
conceivable,  with  our  present  chemical  knowledge,  how  they  can 
give  rise  to  urea  in  any  way  other  than  by  being  broken  down 
into  an  ammonia  stage  and  a  subsequent  synthesis  of  urea  from 
this  product.  The  synthesis  may  however  involve  any  one  of  the 
three  following  processes.  The  ammonia  may  unite  with  carbonic 
acid  to  form  ammonium  carbonate,  which  is  then  dehydrated  into 
urea  (Schmiedeberg).  Again,  it  may  unite  with  carbamic  acid  to 
form  the  carbamate,  which  again  by  loss  of  one  molecule  of  water 
yields  urea  (Drechsel).^  But  in  the  third  place  the  ammonia 
residues  may  unite  with  some  cyanic  compound  to  form  urea  in 
accordance  with  the  possibilities  indicated  above  (pp.  156,  160) 
(Salkowski  and  Hoppe-Seyler).  The  view  that  some  cyanic  resi- 
dues may  be  involved  in  the  formation  of  urea,  while  at  present 
devoid  of  any  striking  positive  evidence  in  its  support,  is  at  first 
sight  most  attractive,  especially  when  it  is  borne  in  mind  how 
great  the  molecular  energy  of  the  cyanogen  compounds  is,  so  that 
during  their  degradation  in  the  tissues  much  energy  would  be  set 
free.  Pfliiger,^  following  Liebig,  has  called  attention  to  this  great 
molecular  energy  of  the  cyanogen  compounds,  and  has  suggested 
that  the  functional  metabolism  of  protoplasm,  by  which  energy  is 
set  free,  may  be  compared  to  the  conversion  of  the  energetic  un- 
stable cyanogen  compounds  into  the  less  energetic  and  more  stable 
amides.  In  other  words,  ammonium  cyanate  is  a  type  of  living, 
and  urea  of  dead  nitrogen,  and  the  conversion  of  the  former  into 
the  latter  is  an  image  of  the  essential  change  which  takes  place 
when  a  living  proteid  dies. 

If  we  accept  this  view  it  is  perhaps  difficult  to  understand  how 
the  cyanic  compounds,  poisonous  as  they  are  known  I/O  be,  could 
play  a  part  in  the  body.  But  it  is  apparently  the  (CN)  group 
which  confers  on  the  compounds  their  poisonous  properties ;  and 
if  cyanic  acid  be  truly  carbamide  CO  .  NH  this  group  is  non- 
existent in  it,  and  it  has  been  recently  stated  that  cyanuric  acid 
(CO .  NH)3  when  introduced  into  the  body  leads  to  an  increased 
excretion  of  urea.^ 

One  difficulty  in  connection  with  this  view  is  that  as  jet  cyanic 
acid  has  never  been  obtained  by  the  artificial  decomposition  of  pro- 
teids.  But  on  the  other  hand  the  proteids  are  the  chief  and  only 
source  of  the  cyanogen  compounds,  for  which  the   starting-point   is 

1  Cf.  above  sub  sarkosin,  p.  140,  and  carbamic  acid,  p.  151. 

2  Pfliiger's  Arch.  Bd.  x.  (1875),  S.  337. 

^  Coppola,  Rendic.  d.  R.  Ace.  d.  Lincei,  1889,  pp.  378,  668.  Ann.  di  Chim.  e 
difarmac.  (4)  T.  x.  (1889),  p.  3. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.        163 

found  in  ferrocyanide  of  potassium,  prepared  by  fusing  nitrogenous 
animal  refuse  with  potassium  carbonate  and  iron.  There  is  further 
evidence  of  the  existence  in  the  body  of  cyanic  residues,  as  shown  by  the 
exit  from  it  of  sulphoc\'anates  (HCXS),  which  are  found  in  both  saliva 
and  more  particularly  in  urine. -^  The  existence  of  sulphvir  in  these 
salts  suggests  at  once  that  it  arises  from  the  decomposition  of  proteids, 
into  Avhose  composition  sulphur  enters  as  a  constant  and  characteristic 
constituent.  The  formation  of  sulphocyanic  acid  in  the  body  has 
recently  been  investigated,  aud  it  is  worthy  of  note  that  it  is  stated 
to  occur  in  the  urine  only  of  those  animals  which  excrete  their  nitro- 
gen chiefly  in  the  form  of  urea.^ 

The  various  ways  by  whicli  it  has  been  suggested  that  urea  may 
arise  in  the  body  all  imply  that  whatever  be  the  form  in  which 
the  nitrogen  initially  leaves  the  tissues,  the  substance  or  sub- 
stances in  which  it  makes  its  exit  undergo  their  final  (synthetic  ?) 
conversion  in  some  other  organ  of  the  body.  In  the  case  of  leucin 
there  is  distinct  evidence  that  the  conversion  is  effected  in  the 
liver,  and  there  is  increasing  evidence  that  this  organ  is  largely 
concerned  in  the  presumably  synthetic  changes  which  lead  to  the 
formation  of  urea  in  mammals  and  of  uric  acid  in  birds.  Thus 
Schroder  has  shown  that  the  conversion  of  ammonium  carbonate 
into  urea  occurs  in  the  liver,^  and  a  similar  relationship  to  the 
formation  of  uric  acid  in  birds  has  additionally  been  proved.* 
Further  there  are  many  observations  which  show,  Avhen  the  liver 
is  diseased,  a  marked  diminution  in  the  excretion  of  urea,  with  a 
frequently  increased  output  of  ammonia.^  After  extirpation  of 
the  liver  in  bhds  the  urine  contains  not  only  more  ammonia  but 
a  large  amount  of  sarcolactic  acid.^  It  would  be  however  prema- 
ture to  regard  this  fact  as  showing  that  in  birds  uric  acid  is  partly 
formed  by  the  converting  activity  of  the  liver  brought  to  bear 
upon  ammonia  and  lactic  acid.  When  urea  is  given  to  birds  it 
reappears  externally  as  uric  acid,''  but  this  change  is  not  effected 
after  extirpation  of  the  liver. 

Substituted  Ureas.  The  hj'drogen  atoms  of  urea  can  be  replaced 
by  alcohol-  and  acid-radicles.  The  results  are  substituted  ureas  in  the 
first  case,  or  ureides  as  they  are  called  in  the  second,  when  the  hj^dro- 
gen  is  replaced  by  the  radicle  of  an  acid.  Many  of  them  are  called 
acids,  since  the  hydrogen  from  the  amido  group,  if  not  all  replaced  as 
above,  can  be  replaced  by  a  metal.     Thus  the  substitution  of  oxalyl 

1  Munk,  Virchow's  Arch.  Bd.  lxix.  (1877),  S.  354.  Gscheidlen,  Pfliiger's  Arch. 
Bd.  XIV.  (1877),  S.  401. 

2  Bruvlants,  Bull,  de  I'acad.  de  me'd.  de  Belgique,  (4)  T.  il.  (1888),  p.  18  et  seq. 

3  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  xv.  (1882),  S.  364  ;  Bd.  xix.  (1885),  S.  373. 
Cf.  W.  Salomon,  Virchow's  Arch.  Bd.  xcvii.  (1884),  S.  149. 

4  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  xxi.  (1886),  S.  40. 

5  Koster,  Lo  Sperimentale,T.  :s.liv.  (1879),  p.  153.  Hallervorden,  ^rc/i.  /.  e.rp. 
Path.  u.  Pharm.  Bd.  xii.  (1880),  S.  237.  Stadelmann,  Deutsch.  Arch.  f.  klin.  Med. 
Bd.  XXXIII.  (1883),  S.  526. 

6  Minkowski,  loc.  cit.  See  also  Marcuse,  Pfliiger's  Arch.  Bd.  xxxix.  (1886),  S. 
425. 

^  Meyer  u.  Jaffe,  Ber.  d.  d.  chem.  Gesell.  Bd.  x.  (1877),  S.  1930. 


164 


UREA. 


/ 


NH.CO 


(oxalic  acid)  gives  parabanic  acid,   COC  |  ;  of  tartronyl  (tar- 

NH.CO 

tronic   acid),    dialuric   acid,    CO.  /CtlOH;    of    mesoxalyl 

NH.CO 
^NH.  CO  ^ 

(mesoxalic  acid),  alloxan  CO  .  .CO.     These  substances  are 

NH.CO 
interesting  as  being  also  obtained  by  the  artificial  oxidation  of  uric 
acid.     The  close  chemical  relationship  of  urea  to  uric  acid  will  be 
explained  below. 

Uric  acid.    C5H4N4O3. 

The  chief  constituent  of  the  urine  in  birds  and  reptiles;  it 
occurs  only  sparingly  in  this  excretion  in  man  (-2-1  grm.  in  24 
hours)  and  most  mammalia.     It  is  normally  present  in  the  spleen, 


Rapidly  separated. 
Fig.  ^7.    Crystals  of  Uric  Acid. 


Slowl}'  separated. 
(Krukenberg  after  Kiihne.) 


and  traces  of  it  have  been  found  in  the  lungs,  muscles  of  the  heart, 
pancreas,  brain,  and  liver.  Urinary  and  renal  calculi  often  consist 
largely  of  this  substance,  or  its  salts.  In  gout,  accumulations  of 
uric  acid  salts  may  occur  in  various  parts  of  the  body,  more  espe- 
cially at  the  joints,  forming  the  so-called  gouty  concretions. 

It  is  when  pure  a  colourless,  crystalline  powder,  tasteless,  and 
without  odour.  The  crj^stalline  form  is  very  variable,  differing 
according  to  the  concentration  of  the  solution  from  which  the 
crystals  are  obtained,  the  rate  at  which  they  are  formed,  and 
whether  they  are  separated  out  spontaneously  or  by  the  addition 
of  acids  to  either  solutions  of  the  acid  or  to  urine.  Hence  it  is 
extremely  difficult  to  illustrate  them  within  reasonable  limits, 
and  for  figures  of  the  various  possible  forms  some  special  work 
must  be  consulted.^     The  impure  acid  crystallises  much  more 

1  See  Ultzmann  and  K.  B.  Hofmaun,  Atlas  der  Harnsedimente,  Wieii,  1872. 
Also  Funke,  Atlas  d,  physiol.  Chem.  Leipzig,  1858. 


CHEMICAL  BASIS  OF  THE  ANIMAL  BODY. 


165 


The  following  figure  shows  addi- 


readily  than  does  the  purified. 

tionally  some  very  characteristic  forms  in  which  uric  acid  sepa- 
rates out  from  urine  either  spontaneously  or  after  the  condition  of 
hydrochloric  acid. 


Fig.  18.     Crystals  of  Uric  Acid.     (After  Eunke.) 

Uric  acid  is  remarkably  insoluble  in  water  (1  in  14,000  or 
15,000  of  cold  water,  1600  of  boiling).  Ether  and  alcohol  do  not 
dissolve  it  appreciably.  On  the  other  hand,  sulphuric  acid  takes 
it  up  in  the  cold  without  decomposition,  and  it  is  also  readily 
soluble  in  many  salts  of  the  alkalis,  as  in  the  caustic  alkalis 
themselves  ;  ammonia  however  scarcely  dissolves  it,  and  in  this 
respect  it  differs  conveniently  from  cystin.  It  is  fairly  soluble 
in  glycerin,  and  soluble  to  some  extent  in  solutions  of  lithium 
carbonate. 


Fig.  19.     (Krukenberg  after  Kiihne.) 

Urinary  sediment,  showing  chiefly  the  most  usual  form  of  crystals 
of  acid  sodium  urate.  C5H3Na]Sr403. 


166 


UKIC   ACID. 


Salts  of  Uric  acid.  Of  these  the  most  important  are  the  acid 
urates  of  sodium,  potassium,  and  ammonium ;  these  salts  are  fre- 
quently still  called  '  lithates,'  the  term  '  lithic '  acid  being  used 
for  uric  acid.  The  sodium  salt  which  is  the  most  common  con- 
stituent of  many  urinary  sediments  crystallises  in  many  different 
forms,  these  not  being  characteristic,  since  they  are  almost  the 
same  for  the  corresponding  compounds  of  the  other  two  bases.  It 
is  very  sparingly  soluble  in  cold  water  (1  in  1100  or  1200),  more 
soluble  in  hot  (1  in  125).  It  is  the  principal  constituent  of  several 
forms  of  urinary  sediment,  and  composes  a  large  part  of  many 
calculi ;  the  excrement  of  snakes  contains  it  largely.  The  potas- 
sium resembles  the  sodium  salt  very  closely,  as  also  does  the 
compound  with  ammonium ;  the  latter  occurs  generally  in  the 
sediment  from  alkaline  urine. 


Fig.  20.     (Krukenberg  after  Kiihne.) 

Urinary  sediment  from  alkaline  urine.  The  large  crystals  consist 
of  ammonio-magnesium  phosphate  (triple  phosphate,  NH4MgP04  -j- 
6H2O).  A  few  crystals  (octahedra)  of  calcium  oxalate  are  also  shown. 
The  remaining  crystals  represent  the  form  of  acid  ammonium  urate, 
C5H3(NH4)N403.     The  rounded  objects  are  urinary  fungi. 

Preparation.  The  amount  of  uric  acid  in  mammalian  urine  is 
too  small  to  make  it  a  source  of  the  acid.  Crystals  may  however 
be  readily  obtained  from  human  urine  by  adding  to  it  2  —  3  p.  c. 
of  strong  hydrochloric  acid  and  letting  it  stand  for  one  or  two 
days  in  a  cool  place.  The  crystals  form  on  the  sides  of  the  con- 
taining vessel. 

On  the  large  scale  it  is  usually  prepared  from  guano,  or  frbm 
snake's  excrement.  From  the  latter  it  is  obtained  by  boiling  with 
caustic  potash  (1  part  alkali  to  20  of  water)  as  long  as  ammonia 
is  evolved ;  in  the  filtrate  a  precipitate  of  acid  urate  of  potassium 
is  formed  by  passing  a  current  of  carbonic  acid  ;  this  salt  .is  tiien 
washed,  dissolved  in  caustic  potash,  and  decomposed  by  carefully 
filtering  its  solution  into  an  excess  of  dilute  hydrochloric  acid. 


CHEMICAL   BASIS   OF   THE  ANIMAL   BODY.        167 

By  similar  treatment  uric  acid  is  readily  obtained  from  fowl's 
excrement,  a  convenient  source  of  the  acid. 

Identification  of  uric  acid.  The  crystalline  forms  afford  some 
clue,  but  are  so  numerous  that  some  forms  which  may  at  any 
time  present  themselves  are  scarcely  characteristic.  The  rhombic 
tables,  '  dumb-bell,'  and  '  whetstone '  crystals  are  on  the  whole 
most  characteristic. 

i.  Murexid  test.  The  suspected  substance  is  treated  in  a  por- 
celain dish  with  a  few  drops  of  strong  nitric  acid  and  evaporated 
carefully  to  dryness,  by  preference  on  a  water-bath.  The  residue 
thus  obtained  will,  if  uric  acid  is  present,  be  of  a  yellow  or  more 
frequently  red  colour,  which  turns  to  a  brilliant  reddish  purple  on 
exposure  to  the  vapours  of  ammonia.  On  the  subsequent  addition 
of  a  drop  of  caustic  soda  the  colour  is  changed  to  a  reddish  blue. 
This  disappears  on  warming,  whereas  the  similar  colour  obtained 
by  the  above  process  from  guanin  does  not.  This  is  an  important 
means  of  distinguishing  between  the  two  substances. 

The  test  depends  on  the  formation  of  murexid,  which  is  the  acid 
ammonium  salt  of  purpuric  acid,  the  acid  itself  being  unknown  in  the 
free  state.  Uric  acid  is  decomposed  when  heated  with  nitric  acid, 
yielding  alloxan  and  then  alloxantin  ;  by  the  action  of  ammonia  the 
latter  is  converted  into  murexid  (NH4)  C8H4X5O6  ~\-  HgO. 

The  murexid  test  is  so  striking  and  characteristic  that  it  suf- 
fices completely  for  the  identification  of  uric  acid.  The  following 
tests  may  be  applied  in  confirmation  if  required,  but  not  for  the 
purposes  of  initial  detection. 

ii.  Schif's  reaction.'^  The  substance  is  dissolved  in  sodium 
carbonate,  and  a  drop  is  then  placed  on  filter  paper  previously 
moistened  with  nitrate  of  silver.  A  yellow  or  almost  black 
colouration,  due  to  the  formation  of  metallic  silver  by  reduction 
of  its  nitrate,  is  at  once  obtained. 

iii.  When  a  solution  of  uric  acid  in  caustic  soda  is  boiled  with 
a  small  amount  of  Fehling's  fluid,  reduction  occurs  with  produc- 
tion of  a  greyish  precipitate  of  urate  of  cuprous  oxide.  If  the  cop- 
per salt  is  in  excess  red  cuprous  acid  is  obtained. 

Estimation  of  uric  acid  in  solutions  {urine).  The  accurate 
quantitative  determination  of  uric  acid  is  a  matter  of  some  dif- 
ficulty ;  for  details  some  standard  works  (quoted  sub  urea) 
should  be  consulted.  It  will  suffice  to  indicate  here  the  princi- 
ples of  the  more  usually  employed  methods. 

i.    Salkowsld-Liidioig  metliod?     When  an  ammoniacal  solution 

1  Ann.    d.  Chem.  u.  Pharm.  Bd.  Cix.   (1859),  S.  65. 

2  Ludwig,  Wien.  med.  Jahrb.  1884,  S.  597.  Cf.  Camerer,  Zt.  f.  Biol.  Bd.  xxvii. 
(1890),  S.  153. 


168  URIC   ACID. 

of  nitrate  of  silver  is  added  to  a  solution  of  uric  acid,  to  which  an 
ammoniacal  mixture  of  magnesium  chloride  and  ammonium  chlo- 
ride has  been  previously  added,  the  uric  acid  is  precipitated  as  a 
magnesio-silver  salt.  This  is  collected,  washed,  and  decomposed 
by  sodium  or  potassium  hydrosulphide,  whereupon  the  uric  acid 
passes  again  into  solution  as  a  urate  of  the  alkali.  On  the  addi- 
tion of  an  excess  of  hydrochloric  acid  to  this  solution  the  urate  is 
decomposed,  uric  acid  separates  out  and  is  collected  and  weighed. 

ii.  Haycraft's  method}  When  uric  acid  is  precipitated  by  am- 
moniacal solution  of  nitrate  of  silver  in  presence  of  the  ammonio- 
magnesic  mixture  as  above  described  the  precipitate  is  stated  to 
contain  one  atom  of  silver  to  each  molecule  of  uric  acid.  The 
uric  acid  is  hence  determined  by  dissolving  the  precipitate  in  nitric 
acid,  in  which  solution  the  silver  is  then  estimated  volumetrically 
with  a  standard  solution  of  potassium  sulphocyanate.^ 

Chemical  constitution  of  uric  acid.  Notwithstanding  the  fre- 
quent and  careful  investigation  of  uric  acid  and  of  the  extremely 
numerous  products  of  its  decomposition,  its  constitution  has  until 
recently  been  a  matter  chiefly  of  surmise  and  conjecture,  and 
many  constitutional  formulae  have  been  assigned  to  it.  When 
uric  acid  is  treated  with  concentrated  hydriodic  acid  at  160-170° 
it  is  decomposed  into  glycin,  ammonia,  and  carbonic  anhydride 

C5H4N4O3  +  5H2O  =  CH2(NH2)  .  COOH  .  +  3CO2  +  3NH3. 

By  reversing  this  decomposition  as  it  were,  namely  by  fusing  to- 
gether at  200-230°  glycin  and  urea,  uric  acid  was  for  the  first 
time  obtained  artificially  ;  ^  when  sarkosin  is  used  instead  of  urea 
methyl-uric  acid  is  obtained.  Uric  acid  has  also  been  prepared 
by  fusing  together  trichlor-lactamide  or  trichlor-acetic  acid  and 
urea.*  The  high  temperatures  at  which  the  above  reactions  were 
conducted  and  the  uncertainty  as  to  the  nature  of  the  products 
intermediate  between  the  reagents  and  the  finally  formed  uric 
acid  precluded  them  from  being  regarded  as  syntheses  in  the  strict 
sense  of  the  word.  A  true  synthesis  of  uric  acid  has  been  recently 
discovered  by  Behrend  and  Roosen,^  from  which  it  appears  that  the 
constitutional  formula  first  assigned  to  the  acid  by  Medicus,^  is  a 
true  representation  of  its  constitution.  This  view  had  been  pre- 
viously stated  by  E.  Fischer  as  a  result  of  his  analytical  investiga- 
tions of  uric  acid.^ 

^  Brit.  Med.  Jl.  1885,  p.  1100.  .Tl.  of  Anat.  and  Physiol.  Vol.  xx.  p  69.5.-  Zt.  f. 
anal.  Chem.  Bd.  xxv.  (1885),  S.  165.     Zt.f.  physiol.  Chem.  Bd.  xv.  (1891),  S.  436. 

2  Volhard,  Jn.  f.  pr.  Chem.  (2)  Bd.  ix.  (1874),  S.  217. 

^  Horbaczewski,  Monatsh.f.  Chem.  Bd.  iii.  (1882),  S.  796.  Ber.  d.  deutsch.  chem.. 
Gesell.  Jahrg.  (1882),  S.  2678. 

*  Horbaczewski,  Monatsh.f.  Chem.  Bd.  vi.  (1885),  S.  356;  Bd.  viii.  (1887),  Sn. 
201,  584. 

5  Ann.  d.  Chem.  u.  Pharm.  Bd.  CCLi.  (1889),  S.  235. 

6  Ibid.  Bd.  CLXxv.  (1875),  S.  230. 

'  Ber.  d.  deutsch.  chem.  Gesell.  1884,  Sn.  328,  1785. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        169 


NH  — CO 


Uric  acid. 


CO       C  — NH 

I  II 

NH— C  — NH 


)C0. 


An  inspection  of  the  above  formula  shows  at  once  that  uric 
acid  contains  the  residues  of  two  molecules  of  urea.  This  cor- 
responds to  the  fact  that  nearly  all  the  possible  decompositions  of 
uric  acid  yield  either  a  molecule  of  urea  along  with  the  more  spe- 
cific product  of  the  decomposition,  frequently  itself  a  derivative  of 
urea,  or  else  some  substance  which  can  by  further  change  be  de- 
composed into  urea  and  some  other  product  which  is  as  before 
frequently  a  derivative  of  urea.  The  close  chemical  relationship 
of  urea  and  uric  acid  is  thus  clearly  shown,  and  may  be  further 
emphasized  by  the  following  reactions,  which  illustrate  and 
amplify  at  the  same  time  the  general  statement  which  has 
just  been  made. 

The  decomposition  of  uric  acid  takes  place  in  two  stages,  yieldr 
ing  two  series  of  products,  of  which  one  is  headed  by  alloxan 
and  the  other  by  allantoin;  from  these  two  substances  respec- 
tively the  other  members  of  each  series  are  derived  by  subsequent 
decomposition. 

1.     Alloxan  series. 

By  careful  oxidation  with  nitric  acid  uric  acid  is  decomposed 
into  a  molecule  of  alloxan  and  one  of  urea. 


NH  — CO 


NH  — CO 


CO        C  —  NH  CO 

I        II  )co  I 

NH— C  — NH  -fH^O+O^NH 


CO      NH 


2\ 


;co. 


CO  +  NHo' 

Alloxan  is  itself  a  substituted  urea  or  ureide  (antea,  p.  164), 
viz.  mesoxalyl-urea,  and  by  oxidation  can  be  further  converted 
into  parabanic  acid   (oxalyl-urea)  and  carbonic  anhydride. 

NH  —  CO  NH  —  CO 


CO       CO 


CO 


NH  — CO  +  0=NH  — CO  +  CO2. 

By  heating  with  alkalis  parabanic  acid  is  hydrated  and  yields 
oxaluric  acid. 

NH  —  CO  NH  —  CO 


CO 


CO 


NH  —  CO  +  H2O  =  NH2      CO .  OH. 


170 


UEIC  ACID. 


The  latter  by  prolonged  boiling  with  water  is  converted  into 
urea  and  oxalic  acid. 


NH  — CO 

i 

CO 


NH 


?\ 


CO 


CO.  OH 


NH2      C0.0H  +  H20  =  NH„  +CO.OH 

2.     Allantoin  sei^ies. 

By  oxidation  with  potassium  permanganate  uric  acid  is  decom- 
posed into  allantoin  and  carbonic  anhydride. 

NH  — CO  NH  — CO       NH2 


CO 

I 
NH 


C  — NH 

11  ^co 

.C-NH-" 


\, 


CO 


CO 


+  H20  +  0  =  NH  — CH  — NH  +  CO2. 

When  allantoin  is  boiled  with  nitric  acid  it  is  hydrated  and 
decomposes  into  a  molecule  of  urea  and  one  of  allanturic  acid. 

NH  — CO      NH2  NH  — CO 


CO 

I 
NH 


CO 


CO 


ch-nh+h,o==co(™^^_^^Ih. 


CH(OH). 

Allanturic  acid  is  itself  a  substituted  urea,  viz.  glyoxyl-urea,  and 
may  be  converted  into  parabanic  and  hydantoic  acids. 

NH  — CO  NH  — CO      NH2         CO.  OH 


2  CO 


CO 


CO 


NH  —  CH  (OH)  =  NH  —  CO  +  NH CH2. 

Of  these  two  acids  the  parabanic  may  as  before  be  converted 
into  oxalic  acid  and  urea,  and  hydantoic  acid  is  a  derivative,  by 
simple  hydration,  of  hydantoin,  which  is  itself  a  substituted  urea, 
viz.  glycolyl-urea,  containing  a  residue  of  glycolic  acid,  [CH2(0H). 
COOH]. 

NH  — CO  NH,  CO.  OH 


CO 


CO 


NH  —  CH2  (Hydantoin)  +  H2O  =  NH CHj.  (Hydantoic  acid. ) 

The  above  reactions  and  decompositions  show  clearly  how  close 
is  the  chemical  relationship  of  urea  and  uric  acid,  and  the  connec- 
tion is  still  more  evident  when  it  can  be  shown  that  many  of  the 
products  described  above  as  obtained  during  the  decomposition  of 
uric  acid,  viz.  the  ureides,  can   be  prepared  from  urea  directly. 


CHEMICAL  BASIS    OF   THE   ANIMAL   BODY.        171 

Thus  parabanic  acid  (oxalyl-urea)  is  readily  formed  by  the  action 
of  phosphorus  oxychloride  on  a  mixture  of  urea  and  oxalic  acid : 

NH— CO 

NH2      CO .  OH  CO 

C0<  I  =      I 

NH„  +  CO  .  OH  NH  —  CO  +  2  H2O. 

When  the  close  chemical  relationship  of  urea  to  uric  acid  is 
taken  into  account,  the  statement  that  those  substances  which 
when  introduced  into  the  body  of  a  mammal-  lead  to  an  increased 
excretion  of  urea,  when  introduced  into  the  organism  of  birds  are 
converted  into  uric  acid,^  needs  excite  no  surprise.  There  is  fur- 
ther distinct  evidence,  already  referred  to  under  urea,  that  the 
conversion  is  affected  in  the  liver.^  We  know  nothing  as  yet  as 
to  the  cause  of  the  slight  divergence  of  metabolism  which  leads 
to  the  preponderating  formation  of  urea  in  mammals  and  of  uric 
acid  in  birds  and  reptiles.  It  is  certainly  not  due,  as  some  have 
supposed,  to  insufficient  oxidation  in  the  latter,  since  the  excretion 
of  uric  acid  is  not  increased  in  mammals  by  artificial  disturbance 
of  the  respiratory  interchange,^  and  it  is  exactly  in  birds  that  the 
most  active  oxidational  changes,  as  shown  by  their  higher  tem- 
perature, is  observed.  Bearing  in  mind  how  readily  uric  acid 
yields  urea  as  one  product  of  its  oxidational  decomposition,  it  has 
been  supposed  that  a  good  deal  more  uric  acid  is  formed  in  the 
mammalian  body  than  is  excreted  in  the  urine.  In  support  of 
this  view  it  may  be  pointed  out  that  uric  acid  when  introduced 
into  mammals  is  largely  excreted  as  urea,  and  that  some  of  the 
known  products  of  the  artificial  oxidation  of  uric  acid  are  occa- 
sionally found  in  their  urine,  e.g.  oxalic  acid,  oxaluric  acid 
(hydrated  parabanic  acid),  and  allantoin.*  The  latter  substance 
is  apparently  increased  (?)  by  the  administration  of  uric  acid.^ 

3.     Oxaluric  acid.     C3H4]Sr204.     (Hydrated  parabanic  acid.) 

Occurs  in  minute  traces  in  normal  urine,  from  which  it  is  ex- 
tracted by  filtering  a  large  quantity  of  urine  very  slowly  through 
a  relatively  small  amount  of  animal  charcoal.  The  charcoal  after 
being  washed  with  distilled  water  is  extracted  with  boiling  alcohol, 
to  which  it  yields  the  oxaluric  acid  as  an  ammonium  salt.  The 
free  acid  is  a  white  crystalline  powder,  not  very  soluble  in  water : 
its  alkaline  salts  are  readily  soluble.^ 

^  For  literature  see  Bunge,  Physiol,  path.  Chemistry,  p.  341.  Horbaczewski, 
Monatshft.  f.  Chem.  Bd.  x.  (1889),  S.'624.  Sitzb.  d.  Wieri.Akad.  Bd.  xcviii.  (1889), 
3  Abth.S.'sOl. 

2  See  also  von  Schroder,  .4?-c^. /.  Physiol.  1880.  Suppl.-Bd.  S.  113.  Ludwig's 
Festschrift,  1887,  S.  98. 

2  Senator,  Virchow's  Arch.  Bd.  xlii.  (1868),  S.  35. 

*  Salkowski  u.  Leube,  Die  Lehre  vom  Ham  (1882),  S.  100. 

5  Salkowski,  Ber.  d.  d.  chem.  Gesell.  1876,  S.  719,  1878,  S.  500. 

^  For  details  see  Hoppe-Seyler,  Phys.-path.  Anal.  1832,  S.  159.  Neubauer  u. 
Vogel,  Harnanalyse,  1890,  S.  239. 


172 


ALLANTOIN. 


4.     Allantoin.    C4H6N4O3.     (Diureide  of  glyoxylic  acid.) 

The  characteristic  constituent  of  the  allantoic  fluid,  more  espe- 
cially of  the  calf,  as  also  in  foetal  urine  and  amniotic  fluid ;  it  occurs 
also  in  the  urine  of  many  animals  for  a  short  period  after  their 
birth.  Traces  of  it  are  sometimes  detected  in  this  excretion  at  a 
later  date.  It  is  obtained  in  urine  after  the  internal  administra- 
tion of  uric  acid.i  It  has  also  been  found  in  vegetable  tissues.^ 
It  crystallises  in  small,  shining,  colourless,  hexagonal  prisms. 
They  are  soluble  in  160  parts  of  cold  water,  more  soluble  in  hot, 
insoluble  in  cold  alcohol  and  ether,  soluble  in  hot  alcohol.  Car- 
bonates of  the  alkalis  dissolve  them,  and  compounds  may  be 
formed  of  allantoin  with  metals  but  not  with  acids.  The  salts 
with  silver  and  mercury  are  important  as  providing  a  means  of 
separating  allantoin  from  its  solutions. 


Fig.  21.     Crystals  from  concentrated  Urine  of  Calf.     (After  Kiihne.) 


The  large  central  crystal  composed  of  an  aggregation  of  small  prisms 
is  allantoin:  those  below  it  are  crystals  of  kreatin,  kreatinin  and  oxa- 
late of  lime.  The  large  prisms  in  the  upper  part  of  the  figure  consist 
of  magnesium  phosphate. 

Allantoin  gives  no  reactions  which  are  sufficiently  striking  to 
admit  of  its  detection  in  urine  or  other  fluids ;  it  must  therefore 
in  all  cases  first  be  separated  out  and  then  examined.  The  separa- 
tion may  be  effected  in  several  ways,  of  which  those  more  usually 
employed  consist  in  its  precipitation  with  nitrate  of  mercury  or 
silver.'^  From  the  urine  of  calves  or  from  their  allantoic  fluid, 
allantoin  may  usually  be  obtained  in  crystals  by  mere  concentra- 
tion and  subsequent  standing  till  crystallisation  occurs. 

1  Salkowski,  he.  cit. 

2  Schulze  u.  Barbieri,  Jn.  f.  pr.  Chem.  Bd.  xxv.  (1882),  S.  145.  Schulze  u. 
Bosshard,  Zt.  f.  physiol.  Chem.Bd.  ix.  (1885),  S.  420. 

3  For  details  see  Hoppe-Seyler,  loc.  cit.  S.  162.  Neubauer  u.  Vogel,  loc.  cit. 
S.  222. 


CHEMICAL  BASIS   OF   THE  ANIMAL   BODY.        173 


Preparation.  Allantoin  may  be  easily  obtained  by  the  careful 
oxidation  of  uric  acid  with  potassium  permanganate.^  It  may 
also  be  synthetised  by  prolonged  heating  to  100°  of  a  mixture  of 
giyoxylic  acid  and  urea,^  or  of  the  latter  substance  with  mesoxalic 
acid.^ 

As  prepared  artificially  it  crystallises  readily  in  large  prismatic 
hexagonal  crystals. 


Fig.  22. 


Crystals  of  Allantoin  prepared  by  the  oxidation 
OF  Uric  Acid.     (After  Kiihne.) 


In  addition  to  the  crystalline  form  and  precipitability  with 
nitrates  of  mercury  and  silver,  allantoin  is  further  characterised 
by  yielding  Schiff's  reaction  with  furfurol  (see  above,  p.  158,  sub 
urea),  but  less  readily  and  with  less  intense  colouration  than  does 
urea.     It  also  reduces  Fehling's  fluid  on  prolonged  boiling. 

The  Xanthiis^  Group.* 

This  group  comprises  a  number  of  substances  closely  related  to 
uric  acid  and  to  each  other.  Some  of  them  occur  in  small  amounts 
in  the  tissues  (muscles)  and  excretions  (urine)  of  the  body  and  are 
to  be  regarded  as  being,  like  urea  and  uric  acid,  typical  products 
of  the  downward  destructive  metabolism  of  proteids.  Some  of 
them  are  closely  related  to  certain  alkaloids  which  occur  in  plants 
(theobromin  and  caffein),  and  which  probably  play  some  not  unim- 
portant part  in  the  nutritional  changes  of  the  animal  body,  since 
they  are  constantly  consumed,  in  some  form  or  other,  by  the  larger 
part  of  the  human  race.  This  relationship  of  the  xanthin-bodies 
to  certain  vegetable  alkaloids  is    further  interesting  when  it  is 

1  Claus,  Ber.  d.  d.  chem.  Gesell.  Bd.  vii.  1874,  S.  227. 

2  Grimaux,  Compt.  Rend.  T.  83  (1876),  p.  62. 

3  Michael,  Ayner.  Chem.  Jl.  Vol.  v.  (1883),  p.  198. 

*  For  a  full  statement  of  the  general  reactions  of  this  group,  and  the  methods  for 
their  separation  and  discrimination,  see  Neubauer  u.  Vogel,  Analyse  des  Hams, 
1890.     Sec.  200—219. 


174 


XANTHIN. 


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CHEMICAL  BASIS   OF   THE  ANIMAL   BODY.        175 

remembered  that  the  latter  are  regarded  by  plant-physiologists  as 
waste-products  of  the  vegetable  organism,  and  are  thus  found 
chiefly  in  those  parts  of  the  plant  which  are  on  their  way  to  re- 
moval, viz.  the  bark,  leaves,  and  seeds. 

Many  members  of  this  group  are  both  derivable  from  and  con- 
vertible into  other  members  of  the  group  by  simple  chemical 
processes,  but  this  relationship  of  the  one  to  the  other  will  be 
more  fully  appreciated  by  consideration  of  the  properties  and 
reactions  of  the  separate  substances.  Their  relationships  to  uric 
acid  and  each  other  are  in  many  cases  indicated  by  comparison 
of  their  formulae. 


1.  Xanthin.    C5H4N4O2. 


NH  — CH 


CO 


C— NH 


NH  —  C  =  N 


—  AT     / 


CO  (Fischer)i. 


First  discovered  in  a  urinary  calculus,  and  called  xanthic  oxide. 
More  recently  it  has  been  found  as  a  normal,  though  very  scanty, 
constituent  of  urine,  muscles,  and  several  other  tissues,  such  as 
the  liver,  spleen,  thymus,  brain-substance,  &c.  It  occurs  in  larger 
quantities,  together  with  hypoxanthin,  in  '  extract  of  meat,'  and 
is  also  found  in  traces  in  vegetable  tissues,  —  lupins,  malt-seed- 


FiG.  23.     Xanthin  hydrochloride, 
C5H4N4O2 .  HCl.     (Kiihne.) 


Fig  24.     Xanthin  nitrate, 
C5H4N4O2 .  HNO3.     (Kiihne.) 


lings,  and  tea.  In  nearly  all  cases  it  is  accompanied  by  hypo- 
xanthin. The  amount  which  is  present  in  any  of  the  above 
tissues  and  fluids  is  so  small  that  none  of  them,  except  perhaps 
the  extract  of  meat,  affords  a  convenient  source  for  its  prepara- 
tion.2  To  obtain  it  in  quantity  guanin  is  treated  with  nitrous 
acid,^  and  the  nitro-product  thus  obtained  is  reduced  in  ammonia- 
cal  solution  with  ferrous  sulphate.  It  may  also  be  prepared 
artificially  from  hydrocyanic  acid  and  water  in  presence  of  acetic 
acid.*  When  pure  it  is  a  colourless  powder,  requiring  about 
14,000  parts  of  water  for  its  solution  at  ordinary  temperatures, 

1  (i)  Ber.  d.  d.  chem.  Gesell.  1882,  S.  453.  (ii)  Ann.  d.  Chem.  u.  Pharm.  Bd. 
ccxv.  (1882),  S.  253. 

2  For  its  separation  from  urine  see  Neubauer,  Zt.f.  anal.  Chem.  Bd.  vii.  (1868), 
S.  398  From  muscle-extract,  see  Stadeler,  Ann.  d.  Chem.  u.  Pharm.  Bd.  cxvi, 
.(I860),  S.  102.     Neubauer,  Zt.f.  anal.  Chem.  Bde.  ii.  (1863),  S.  26,  vi.  (1867),  S.  33. 

"  Fischer,  loc.  cit.  (ii). 

*  Gautier,  Compt.  Rend.  T.  98  (1884),  1523. 


176  XANTHIN. 

and  1400  at  100°.  Insoluble  in  alcohol  and  in  ether,  it  dissolves 
readily  in  dilute  acids  and  alkalis  (characteristically  in  ammonia) 
forming  crystallisable  compounds. 

Beactions.  The  discrimination  of  members  of  the  xanthin 
group  is  not  easy,  since  from  their  close  relationship  they  yield 
many  reactions  in  common.  The  following  are  characteristic  of 
xanthin. 

i.  WeideVs  reaction}  The  substance  is  warmed  with  freshly 
prepared  chlorine-water  and  a  trace  of  nitric  acid  as  long  as  any 
gas  is  evolved :  it  is  then  carefully  evaporated  to  dryness  and,  if 
xanthin  is  present,  the  residue  turns  pink  or  purplish-red  on  the 
access  of  ammonia  fumes.  Carnin  gives  a  similar  colouration  if 
but  little  chlorine-water  is  used,  while  guanin  and  adenin  do 
not. 

ii.  Hoppe-Seyler's  reaction.  When  xanthin  is  introduced  into 
some  caustic  soda  with  which  some  chloride  of  lime  has  been 
mixed,  each  particle  of  the  substance  surrounds  itself  with  a  dark 
green  ring  which  speedily  turns  brown  and  then  disappears. 


Fig.  25.     Crystals  of  Xanthin  silver-nitrate,  C5H4N4O2 .  AgNOg. 
(Krukenberg  after  Kiihne.) 

iii.  Strecker's  test?  When  evaporated  to  dryness  on  porcelain 
with  nitric  acid  a  yellow  residue  is  obtained  which  turns  reddish- 
yellow  on  the  addition  of  caustic  soda  or  potash  (not  of  ammonia), 
and  reddish-violet  on  subsequent  warming.  Distinctive  from 
uric  acid. 

iv.  Xanthin  is  more  readily  soluble  in  ammonia  than  is  uric 
acid. 

V.  Xanthin  yields  in  solution  in  dilute  nitric  acid  a  character- 
istic crystalline  compound  with  nitrate  of  silver,  which  differs 
from  the  similar  compound  of  hypoxanthin  both  in  the  forms 
which  it  presents  and  in  its  greater  solubility  in  nitric  acid  of  sp. 
gr.  1-1  at  100°.  It  is  therefore  used  as  a  means  of  separating 
xanthin  and  hypoxanthin. 

1  Ann.  d.  Cliem.  u.  Pharm.  Bd.  clviii.  (1871),  S.  365.  This  reaction  was  given 
by  its  author  for  hypoxanthin,  but  apparently  in  error.  Cf.  Kossel,  Zt.  f.  physiol. 
Chem.  Bd.  vi.  (1882),  S.  426.     Salomon,  Ber.  d.  d.  chem.  Gesell.  1883,  S.  198. 

-  Ann.  d.  Chem.  u.  Pharm.  Bd.  cviii.  (1858),  S.  146. 


CHEMICAL  BASIS   OF   THE   ANIMAL   BODY.        177 

vi.  The  compound  of  xanthin  with  hydrochloric  acid  is  far 
less  soluble  in  water  than  are  the  similar  compounds  of  hypoxan- 
thin  and  guanin,  and  hence  affords  a  further  means  of  separating 
these  bases. 

By  treatment  with  hydrochloric  acid  and  potassium  chlorate 
xanthin  is  converted  into  alloxan  and  urea  (Fischer). 

The  older  and  frequently  repeated  statements  that  xanthin  and 
hypoxanthin  can  he  obtained  from  uric  acid  by  the  action  of  sodium- 
amalgam,  as  also  that  hypoxanthin  can  be  converted  into  xanthin  by 
treatment  with  nitric  acid,  have  recently  been  shown  to  be  erroneous. 
Notwithstanding  the  similarity  of  their  composition  these  three  sub- 
stances are  incapable  of  interconversion.''^ 

2.  Heteroxanthin.    CeHsN^Os  (Methyl-xanthin?). 

This  substance  occurs  in  minute  quantities  in  the  normal  urine 
of  man  ^  and  the  dog,^  along  with  xanthin  and  hypoxanthin  and 
another  closely  allied  xanthiu-base,  paraxanthin.  It  occurs  in 
larger  amount  in  the  urine  of  leukhaemic  patients.  It  is  crystal- 
line, but  not  very  characteristically  so,  is  soluble  with  difficulty 
in  cold  water,  much  more  soluble  in  hot  water,  is  insoluble  in 
alcohol  and  in  ether.  It  may,  as  also  may  paraxanthin,  be  separated 
from  other  xanthin-bases  by  taking  advantage  of  the  relatively 
slight  solubility  of  its  sodium  salt  in  caustic  soda.  It  also  yields 
with  hydrochloric  acid  a  relatively  insoluble  salt  which  crystal- 
lises readily,  whereas  the  corresponding  salt  of  paraxanthin  is 
readily  soluble.  They  may  by  this  means  be  separated  the  one 
from  the  other. 

Heteroxanthin  does  not  give  the  ordinary  reaction  for  xanthin 
with  nitric  acid  and  caustic  soda,  but  yields  a  brilliant  colouration 
on  the  application  of  Weidel's  test  (see  sub  xanthin).  Like  the 
other  xanthin-bases  it  gives  an  insoluble  salt  with  an  ammoniacal 
solution  of  nitrate  of  silver. 

3.  Paraxanthin.  C7H8N4O2  (Dimethylxanthin  ?)  Isomeride  of 
Theobromin. 

Like  heteroxanthin  it  occurs  in  very  small  amounts  in  urine.^ 
It  is  soluble  with  difficulty  in  cold  water,  but  is  more  soluble 
than  xanthin ;  is  much  more  soluble  in  hot  water,  insoluble  in 
alcohol  and  in  ether.  It  crystallises  readily  in  characteristic  flat, 
somewhat  irregular,  six-sided  tables  when  its  solutions  are  slowly 
evaporated,  or  in  needles  if  rapidly.    It  forms,  as  do  the  preceding 

1  Kossel,  Zt.  f.  j^hijsiol  Cheiii.  Bd.  vi.  (1882),  S.  428.  Fischer,  Ber.  d.  d.  ckem. 
Gesell.  1884,  S.  328. 

2  Salomon,  Ibid.  1885,  S.  3407. 

3  Salomon,  Zt.f.  plujsiol.  Chem.  Bd.  xi.  (1887),  S.  412. 

*  Thudiclmm,  A7inals  of  ch.  Med.  Vol  i.  (1879),  p.  166.  Salomon,  Ber.  d.  d. 
chem.  Gesell.  1883,  S.  195,  1885,  3406,  Zt.  f.  Min.  Med.  Bd.  vii.  (SuppL-Hft.)  (1884), 
S.  63.     Cf.  Zt.f.  physiol.  Chem.  Bd.  xv.  (1891),  S.  319. 

12 


178  CARNIK 

substances,  a  crystalline  salt  with  nitrate  of  silver ;  this  like  the 
corresponding  compound  of  xanthin  is  soluble  in  strong  nitric  acid 
(sp.  gr.  I'l)  at  100°,  and  may  thus  be  separated  from  hypoxanthin. 
It  may  be  separated  from  xanthin  by  means  of  its  greater  solubility 
in  cold  water,  and  from  heteroxanthin  by  the  difference  in  the  solu- 
bility of  its  salts  with  sodium  and  hydrochloric  acid. 

Paraxanthin  gives  Weidel's  reaction  but  not  the  ordinary 
xanthin  test  with  nitric  acid  and  caustic  soda. 

An  inspection  of  Fischer's  formula  for  xanthin  shows  the  pos- 
sibility of  the  existence  of  at  least  two  isomeric  di-methyl  deriva- 
tives of  this  base  according  to  the  replacement  by  methyl  CH3  of 
the  hydrogen  atoms  in  the  three  NH  groups  which  it  contains. 
Of  these  one  has  for  some  time  been  known  as  theobromin ;  para- 
xanthin is  probably  another  isomer,  and  more  recently  Kossel  has 
described  a  third,  theophyllin.  By  substitution  of  (CH3)  for 
hydrogen  in  the  third  (NH)  group  trimethyl-xanthin  or  caffein 
is  obtained.  In  connection  with  the  isomeric  relationship  of 
paraxanthin  and  theobromin  it  is  of  great  interest  to  observe  that 
the  physiological  action  of  the  two  bases  is  the  same.^ 

4.     Carnin.    C^HgN^Og. 

Closely  allied  in  composition  to  the  preceding  base,  but  as  yet 
of  unknown  constitution,  carnin  occurs  only  as  a  constituent  of 
'  extract  of  meat,'  of  which  it  forms  about  one  per  cent.,^  although 
it  has  been  stated  to  occur  also  in  urine  (?).^ 

It  is  prepared  by  precipitating  extract  of  meat  with  baryta- 
water,  avoiding  all  excess  of  the  precipitant.  The  filtrate  from 
this  is  now  precipitated  with  basic  acetate  of  lead,  which  carries 
down  all  the  carnin.  This  precipitate  is  repeatedly  boiled  with 
water  which  dissolves  out  the  lead  salt  of  carnin,  which  is  then 
decomposed  by  sulphuretted  hydrogen,  and  the  carnin  obtained 
by  concentration  of  the  aqueous  filtrate  from  the  sulphide  of 
lead.* 

It  crystallises  in  white  masses  composed  of  very  small  irregular 
crystals ;  it  is  soluble  with  great  difficulty  in  cold,  readily  soluble 
in  hot  water,  insoluble  in  alcohol  and  in  ether.  It  unites  with 
acids  and  salts  to  form  crystallisable  compounds.  Of  these  the 
more  important  are  the  salts  with  basic  lead  acetate,  soluble  in 
boiling  water,  and  with  nitrate  of  silver,  insoluble  in  strong  nitric 
acid  and  ammonia.  Carnin  gives  Weidel's  reaction  when  only  a 
small  amount  of  chlorine-water  is  employed,  but  the  test  fails  if 
any  excess  is  used. 

Carnin  bears  an  interesting  relationship  to  hypoxanthin,  into 

1  Salomon,  VerJi.  d.  physiol.  Gesell.  Berlin.     Arch.f.  Physiol.  1887,  S.  582. 

2  Weidel,  Ann.  d.  C'hem.  u.  Pharm.  Bd.  clviii.  (1871),  S,  353. 
8  Pouchet,  Journ.  de  Th^rap.  T.  vii.  (1880),  p.  503. 

'*  Krukenberg  u.  "Wagner,  Sitzb.  d.  phys.-med.  Gesell.  Wiirzburg,  1883,  No,  4. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.        179 

which  it  may  be  converted  by  treatment  with  chlorine  or  nitric 
acid,  or  still  more  readily  by  bromine. 

C7H8N4O3  +  Br^  =  C5H4N4O .  HBr  +  CHsBr  +  CO^. 

The  latter  may  be  isolated  from  its  hydrobromic  acid  salt  by 
means  of  caustic  soda. 

5.     Hypoxanthin  or  Sarkin.     CsHilSTiO. 
NH  —  CH 


CO 


C  — N 


^ 


NH  —  C  =  N 


CH  (?). 


Closely  related  to  xanthin  and  usually  occurring  with  it  in  the 
tissues  and  fluids  of  the  body.  Its  constitutional  formula  has  not 
yet  been  definitely  ascertained,  but  it  will  probably  be  found  to 
contain  the  group  N  =  CH  —  N  in  the  place  of  one  urea  residue 
in  xanthin.^  On  this  supposition  three  formulae  are  obviously 
possible,  and  the  correct  one  has  still  to  be  determined.  Hypo- 
xanthin may  be  obtained  from  normal  muscles,  and  hence  is  found 
in  larger  amounts  in  'extract  of  meat.'  It  occurs  also  in  the 
spleen,  liver,  and  medulla  of  bones,  and  in  considerable  quantity 
in  the  blood  ^  and  urine  ^  of  leukhsemic  patients  ;  also  in  normal 
urine  *  and  in  vegetable  tissues  —  lupins,^  malt-seedlings,  and 
tea.^ 


Fig.  26.     Hypoxanthin-silver-nitrate,  O5H4N4O  .  AgNOs. 
(Krukenberg  after  Kiihne.) 

It  is  obtained  from  fluids  or  tissue  extracts  by  means  of  the 
processes  already  mentioned  for  the  extraction  of  xanthin,  and 
is  separated  from  the  latter  by  taking  advantage  of  the  slighter 

1  Fischer,  Ber.  d.  d.  chem.  Gesell.  1882,  S.  455. 

2  Kossel,  Zt.  f.  physiol.  Chem.  Bd.  v.  (1881),  S.  267. 

3  Stadthagen,  Virchow's  Arch.  Bd.  cix.  (1877),  S.  390. 

*  G.  Salomon,  Zt.  f.  physiol.  Chem.  Salkowski,  Virchow's -4 jtA.  Bd.  l.  (1870), 
S.  195. 

5  Salomon,  Verhand.  d.  physiol.  Gesell.  Nov.  12,  1880.  Arch.  f.  Physiol.  1881,  S. 
166. 

6  Baginsky,  Zt.f.  physiol.  Chem.  Bd.  viii.  (1883—4),  S.  395. 


180 


HYPOXANTHIN. 


solubility  of  its  salt  witli  nitrate  of  silver  in  boiling  nitric  acid 
(sp.  gr.  ll).     The  crystalline  form  of  this  salt  is  characteristic. 

It  also  yields  crystalline  salts  with  nitric  and  hydrochloric 
acids. 

Hypoxanthin  is  soluble  in  300  parts  of  cold  and  78  of  boiling 
water,  insoluble  in  cold  alcohol  and  in  ether,  soluble  in  900  parts 
of  boiling  alcohol.  It  does  not  yield  either  Weidel's  reaction  or 
the  reaction  with  nitric  acid  and  caustic  soda  so  characteristic  of 
the   other  xanthin   bases.     It   gives  no  green  colouration  with 


Fig.  27.     Hypoxanthin-nitrate,  C5H4N4O  .  HNO3.     (Kiihne. 


Fig.  28.    Hypoxanthin-hydrochlokide,  Cj;H4N40 .  HCl.     (Kiihue.) 


caustic  soda  and  chloride  of  lime  such  as  xanthin  does  (Hoppe- 
Seyler's  reaction),  but  after  treatment  with  hydrochloric  acid 
and  zinc,  it  yields  a  ruby-red  colouration  on  the  addition  of  an 
excess  of  caustic  soda  (Kossel).  In  this  reaction  it  resembles 
adenin. 

During  the  putrefactive  decomposition  of  proteids  (fibrin)  or 
by  the  action  of  boiling  water,  dilute  acids,  or  gastric  and 
pancreatic  enzymes,  hypoxanthin  can  be  obtained  in  minute 
amounts.^  This  was  at  first  regarded  as  evidencing  a  direct 
formation  of  xanthin  bases  from  proteids.  The  researches  of 
Kossel  have  however  shown  that  the  source  of  the  hypoxanthin 
in  the  above  cases  is  probably  the  nuclein  of  the  corpuscles  en- 
tangled in  the  fibrin,  since  he  finds  that,  by  similar  treatment, 

1  Salomon,  Ber.  d.  d.  chem.  Gesell.  1878,  S.  574.  Krause,  Inaiig.-Diss.  Berliu, 
1878.     Chittenden,  Jl.  of  Physiol.  Vol.  n.  (1879),  p.  28. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.        181 

isolated  nuclein  yields  no  inconsiderable  amount  of  hypoxanthin.^ 
The  nuclein  however  from  egg-yolk  does  not  yield  hypoxanthin, 
and  thus  resembles  the  nuclein  derivable  from  casein.^  Although 
the  xanthin-bases  undoubtedly  result  in  the  body  from  the  meta- 
bolism of  nitrogenous  (proteid)  tissues  there  is  as  yet  no  evidence  as 
to  the  manner  in  which  they  can  be  formed  from  true  proteids.^ 
The  genetic  relationship  of  hypoxanthin  to  nuclein  proljably  ac- 
counts for  the  marked  occurrence  of  the  former  in  leukhaemic 
blood. 

Bearing  in  mind  the  close  chemical  relationship  of  uric  acid, 
xanthin,  and  hypoxanthin,  and  regarding  the  xanthin  bases  as 
distinctly  and  typically  products  of  the  downward  metabolism 
of  nitrogenous  tissues,  the  question  at  once  suggests  itself 
whether  in  the  body  there  is  any  antecedental  relationship  be- 
tween these  substances  and  uric  acid  (or  urea).  As  with  kreatin 
(above,  p.  162),  so  with  the  xanthin  bodies,  the  disproportion 
between  the  amount  presumably  arising  in  the  tissues  and  that 
which  is  actually  excreted  makes  it  probable  that  they  are  con- 
verted into  something  else,  uric  acid  (or  urea),  before  leaving  the 
body.  And  in  support  of  this  belief  there  is  a  certain  amount 
of  experimental  evidence  which  was  wanting  in  the  case  of 
kreatin.  It  is  found  that  hypoxanthin  administered  to  a  dog 
does  not  reappear  as  such  externally  in  the  urine,*  and  that  when 
given  to  fowls  it  leads  to  an  increased  excretion  of  uric  acid 
amounting  to  some  60  p.  c.  of  the  hypoxanthin  employed.'^  Since 
the  latter  result  is  obtained  in  fowls  with  extirpated  livers,  it  ap- 
pears that  the  conversion  is  not  effected  in  this  organ,  although  it 
is  known  that  normally  no  inconsiderable  portion  of  the  uric  acid 
is  formed  in  their  liver. 

6.     Adenin.     C5H5N5. 

This  base  was  obtained  by  Kossel  ^  during  the  treatment  of 
pancreatic  tissue  for  the  preparation  of  hypoxanthin.  It  bears 
the  same  relationship  to  the  latter  that  guanin  does  to  xanthin, 
and  can  similarly  be  converted  into  hypoxanthin  by  the  action  of 
nitrous  acid.     It  is  stated  to  have  been  found  in  urine." 

1  Zt.f.  pJiysiol.  Chem.  Bde.  in.  (1879),  S.  284,  iv.  290,  v.  152,  267,  vi.  423,  vii.  7. 
Cf.  Low,  Pfliiger's  Arch.  Bd.  xxii.  (1880),  S.  62. 

2  Kossel,  Verhandl.  d.  physiol.  GeselL,  Arch.  f.  Physiol.  1885,  S.  346. 

3  Cf.  Drechsel,  Ber.  d.  d.  chem.  GeselL  1880,'  S.  240.  But  see  also  Salomon,  Ibid. 
S.  1160. 

*  Baginsky,  Zt.f.  physiol.  Chem.  Bd.  viii.  (1884),  S.  397. 

5  Von  Mach,  Arch.  f.  exp.  Path.  u.  Pharm.  Bde.  xxiii.  (1887),  S.  148,  xxiv. 
(1888),  S.  389.     See  also  Stadthagen,  he.  cit.  below. 

6  Ber.  d.  d.  chem.  GeselL  1885,  Sn.  79,  1928,  Zt.  f.  physiol.  Chem.  Bde.  x.  (1886), 
S.  250,  XII.  (1888),  S.  241,  xvi.  (1892),  S.  1.  See  also  Schindler,  Ibid.  xiii.  (1889), 
S.  432.  Gives  directions  for  separation  of  xanthin,  hypoxanthin,  guanin,  and 
adenin.  Thoiss,  Ibid.  Bd.  xiii.  S.  395.  Bruhns,  Ibid.  Bd.  xiv.  (1890),  S.  533. 
KriJger,  Ibid.  Bd.  xvi.  (1892),  S.  160. 

"  Stadthagen,  Virchow's  Arch.  Bd.  cix.  (1887),  S.  390. 


182  GUANIK 

When  pure  it  crystallises  in  needles  from  aqueous  solutions. 
Is  soluble  in  1086  parts  of  cold  water,  readily  in  hot  water,  in- 
soluble in  ether,  slightly  soluble  in  hot  alcohol.  Yields  crystal- 
line compounds  with  acids,  also  with  some  salts.  The  compound 
with  nitrate  of  silver  is  soluble  in  hot  nitric  acid  (sp.  gr.  1-1),  and 
is  thus  separable,  together  with  hypoxanthin,  from  xanthin.  It 
also  yields  a  readily  crystalline  compound  with  picric  acid,  which 
is  very  insoluble  in  cold  water  (1  in  3500)  and  may  be  used 
for  its  quantitative  separation  from  solutions  (Bruhns,  loc.  cit.). 
It  does  not  give  the  ordinary  reactions  characteristic  of  the 
xanthin  bodies,  but  like  hypoxanthin  shows  a  red  colouration  on 
the  addition  of  an  alkali  after  treatment  with  hydrochloric  acid 
and  zinc. 

7.    Guanin.    C5H5N5O.  NH  — CH 

I  II 

NH  =  C  C  —  NH 

I  1  ^CO  (Fisclier).i 

NH  —  C  =  N     ^ 

It  was  first  obtained  from  Peruvian  guano,  which  still  provides 
the  most  convenient  source  for  its  preparation. 

The  guano  is  finely  powdered  and  boiled  with  milk  of  lime  as  long 
as  it  jaelds  a  coloured  filtrate.  The  residue  is  then  repeatedly  ex- 
tracted with  boiling  solution  of  sodium  carbonate;  the  filtrate  on  the 
addition  of  acetic  acid  yields  a  precipitate  of  guanin  and  some  uric  acid, 
from  which  it  is  separated  by  boiling  with  somewhat  dilute  hydro- 
chloric acid.  A  hydrochloride  of  guanin  is  formed  which  is  crystal- 
line, and  from  this  compound  the  guanin  is  separated  by  the  addition 
of  concentrated  ammonia.^ 

Gruanin  is  also  found  in  small  quantities  in  the  pancreas,  liver, 
and  muscle  extract,  and  among  the  products  of  the  action  of  acids 
on  some  nucleins.^  It  may  also  occur  in  urine,  more  especially  of 
pigs,  in  which  case  it  is  also  found  in  many  of  their  tissues ;  *  ad- 
ditionally in  the  retinal  tapetum  of  fishes  and  in  their  scales,^  as 
also  in  the  integument  of  amphibia  and  reptiles,^  and  in  vegetable 
tissues.'' 

It  is  a  white  amorphous  powder,  insoluble  in  water,  alcohol, 
ether,  and  ammonia.     Its  insolubility  in  the  latter  distinguishes 

1  loc.  cit.  (sub  xanthin). 

2  Strecker,  Ann.  d.  Chem.  u.  Pharm.  Bd.  cxviii.  (1861),  S.  152. 

3  Kossel,  Zt.  f.  physiol.  Chem.  Bd.  viii.  (1884),  S.  404. 

*  Pecile,  Ann.  d.  Chem.  u.  Pharm.  Bd.  clxxxiii.  (1876),  S.  141.  Cf.  Salomon, 
Arch.  f.  Physiol.  1884,  S.  17.5.  Arch.  f.  Path.  Anat.  Bd.  xcv.  (1884),  S.  527. 
Virchovv,  Arch.  f.  path.  Anat.  Bde.  xxxV.  (1866),  S.  358,  xxxvi.  S.  147. 

s  Kuhne  u.  Sewall,  Unters.  a.  d.  physiol.  Inst.  Heidelb.  Bd.  in.  (1880),  S.  221. 

6  Ewald  u.  Krukenberg,  Ibid.  Bd.  iv.  Hft.  3.  (1882),  S.  253,  Zt.  f.  Biol.  Bd.  xix. 
(1883),  S.  154. 

■^  Schulze  u.  Bosshard,  Zt.  f.  physiol.  Chem.  Bd.  ix.  (1885),  S.  420. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY. 


183 


it  from  xanthin  and  liypoxanthin.  It  unites  with  acids,  alkalis, 
and  salts  to  form  crystallisable  compounds.  Of  its  compounds 
with  acids  the  most  characteristic  are  those  with  hydrochloric  and 
nitric  acids. 

The  compound  with  nitrate  of  silver  is  extremely  insoluble  in 
strong  boiling  nitric  acid. 


Tig.  29.     Guanin  htdrochloride,  Tig.  30.     Guanin  nitrate, 

C5H5N5O  .  HC1  +  H2O.  (After  Kuhne.)     C5H5N5O  .  HNO3+  H^2^-  (After  Kuhne.) 

Reactions.  By  treatment  with  nitric  acid  and  caustic  soda 
(Strecker's  test)  it  yields  a  colouration  closely  resembling  that 
given  by  xanthin,  but  does  not  respond  to  Weidel's  test  (see 
above,  j).  177). 

Capranica's  reactions}  (i)  A  yellow  crystalline  precipitate  on 
the  addition  of  a  saturated  aqueous  solution  of  picric  acid  to  a 
solution  of  guanin-hydrochloride  ;  insoluble  in  cold  water. 
(ii)  An  orange-coloured  crystalline  precipitate,  very  insoluble 
in  water,  on  the  addition  of  a  concentrated  solution  of  potassium 
chromate.  (iii)  Prismatic  yellowish-brown  crystals  on  the  ad- 
dition of  a  concentrated  solution  of  ferricyanide  of  potassium. 
Xanthin  and  liypoxanthin  when  similarly  treated  do  not  yield 
the  last  two  precipitates. 

By  treatment  with  nitrous  acid  guanin  may  be  readily  con- 
verted into  xanthin.  (Cf.  adenin  into  hypoxanthin  by  similar 
treatment.)  By  oxidation  it  yields  guanidin  NH  :  C  (]SrH2)2,  para- 
banic  acid  (see  above,  p.  171)  and  carbonic  anhydride,  a  decompo- 
sition which  obviously  corresponds  to  the  formula  given  above  for 
guanin. 

C5H5N5O  +  3  .  0  +  H,0  =  NH  :  C(NH2)2  +  CsH^N^Os  +  CO... 

1  Zt.f.  physiol.  Chem.  Bd.  iv.  (1880),  S.  2-33. 


184  GUANIDIN. 

8.     Guanidin.     CN3H5.  NH2 

I 
NH=C 

I 
NH2. 

Although  this  substance  does  not  occur  in  the  free  state  in  any 
tissue  or  fluid  of  the  animal  body,  it  is  of  considerable  interest, 
for  it  has  been  obtained  by  the  direct  oxidation  of  proteids  (p.  161) 
and  may  be  made  to  yield  urea  by  treatment  with  boiling  dilute 
sulphuric  acid  or  baryta  water.  NH  :  C  (NH^s  +  H2O  =  (NHo)^  CO 
+  NH3.  Further,  it  affords  a  connecting  link  between  the  xanthin 
series  and  kreatin  (p.  143),  the  latter  substance  being,  as  already 
stated,  methylguanidin-acetic  acid,  while  guanidin  is  itself  the  chief 
product  of  the  oxidation  of  guanin. 

It  may  be  readily  synthetised  in  several  ways ;  of  these  its 
formation  by  the  action  of  alcoholic  ammonia  on  chlorpicrin  (tri- 
chlornitromethan)  CCI3  (NO2)  or  on  cyanogen  iodide  shows  clearly 
its  constitution.     In  the  first  case 

CCI3  (NO2)  +  3NH3  =  NH :  C  (NH,)^  +  3HC1  +  HKO^. 

In  the  second  CNI  +  3NH3  =  NH  :  C  (NHa)^  +  NHJ,  or  in 
other  words  guanidin  may  be  regarded  as  a  compound  of  cyana- 
mide  and  ammonia  CN .  NH.  +  NH3  =  NH  :  C  (NH^V  The 
relationship  to  kreatin  may  now  be  at  once  made  evident  by 
comparing  the  reaction  just  given  with  that  for  the  synthesis  of 
kreatin  from  cyanamide  and  sarkosin  :  — 

NH. 
CN  .  NH2  +  CH2 .  NH(CH3).  COOH  =  NH :  C  ^ 

^N(CH3).CH2.COOH. 

Xanthin  derivatives. 

The  monomethyl  (?)  derivative  of  xanthin  (heteroxanthin)  has 
already  been  described,  as  also  one  of  the  possible  dimethyl  derivatives, 
viz.  paraxanthin. 

When  the  (silver  or)  lead  salt  of  xanthin  (PbC5H2N402)  is  dried 
and  heated  in  sealed  tubes  at  100°  with  methyl  iodide,  iodide  of  lead 
is  formed  together  with  dimethyl-xanthin.^  The  substance  thus 
obtained  is  identical  with  theobromin,  long  known  as  the  character- 
istic alkaloidal  constituent  of  cocoa-beans,  the  fruit  of  Theobroma 
cacao.  A  third  presumably  dimethyl  derivative  of  xanthin  has  re- 
cently been  described  as  occurring  in  tea,  viz.  theophyllin.^  When 
the  silver  salt  of  theobromin  is  further  treated  as  above  with  methyl 
iodide  it  is  converted  into  methjd-theobromin  or  trimethylxanthin, 
which  is  identical  with  the  vegetable  alkaloid,  long  known  under  the 
synonymous  names  of  theine  or  caffeine,  as  occurring  in  the  leaves  or 
seeds  of  many  plants  such  as  tea  and  coffee,  also  in  the  Brazilian 
'  guarana '  prepared  from  the  fruit  of  Paxdinia  sorhilis,  in  '  mate  '  of 

1  E.  Fischer,  he.  cit.  (sub  xanthin). 

2  Kossel,  Zt.f.  phijsiol.  Chem.  Bd.  xiii.  (1889),  S.  298. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        185 

South  America,  an  infusion  of  the  leaves  of  Ilex  Paraguay ensis,  in 
kola-nuts  used  as  food  in  Central  Africa  (the  fruit  of  SercuUa 
acuminata),  in  South  African  'bush-tea,'  and  in  many  other  plants 
from  which  stimulating  beverages  are  obtained  by  infusion.^  Apart 
from  the  close  chemical  relationship  of  the  alkaloidal  principles  of  the 
above  plants  to  the  nitrogenous  crystalline  '  extractives '  of  muscles, 
it  is  interesting  to  notice  further  that  they  seem  to  bear  the  same 
general  relationship  to  the  organisms  in  which  they  respectively  occur. 
There  can  be  but  little  doubt  that  the  xanthin  bodies  (and  uric  acid) 
are  typically  products  of  the  downward  excretionary  nitrogenous 
metabolism  of  animals.  The  alkaloidal  principles  of  plants,  in  this 
case  theobromin  and  caffeine,  may  be  similarly  regarded  as  excre- 
tionary products  and  are  hence  found  collected  in  those  parts  of  the 
plant  which  are  more  immediately  or  ultimately  cast  off,  viz.  the 
leaves,  seeds,  and  bark.  The  facts  already  stated  render  the  consump- 
tion of  theobromin  and  caffeine  in  some  form  or  other  by  practically 
the  whole  human  race  less  surprising  than  it  might  at  first  sight 
appear.  Their  universal  use  also  indicates  that  they  supply  some 
distinct  want  of  the  economy  which  cannot  as  yet  be  explained  purely 
with  reference  to  their  relationship  to  the  nitrogenous  extractives  of 
animal  tissues,  but  rather  to  the  physiological  effect  their  ingestion 
produces.  In  moderate  doses  they  exert  an  agreeable  stimulating 
action  whereby  the  sensations  of  fatigue  and  drowsiness  are  removed, 
the  body  being  thus  enabled  to  exert  itself  with  less  sense  of  effort 
and  less  initial  stimulus,  and  the  mind  is  more  active,  clear-sighted 
and  resistent  to  the  depressing  effects  of  unpleasant  influences.  There 
is  no  evidence,  as  was  at  one  time  assumed,  that  they  act  in  any  way 
by  reducing  the  activity  of  nitrogenous  metabolism.^  In  the  case  of 
cocoa  and  chocolate  we  have  to  deal  not  merely  with  the  stiiuulating 
effects  of  the  theobromin  they  contain,  but  also  with  the  fact  that 
they  are  of  extreme  nutrient  value,  owing  to  the  large  amount  of  fats 
(50  p.c),  proteids  (12  p.c),  and  carbohydrates  which  enter  into  their 
composition.  The  comparative  physiological  action  of  xanthin,  theo- 
bromin, caffeine,  and  some  of  their  derivatives  have  recently  been 
studied  by  Filehne.^ 

The  Akomatic  Series. 
1.     Benzoic  acid.     CgHs .  COOH. 

This  is  not  found  as  a  normal  constituent  of  the  body.  When 
it  occurs  in  (chiefly  herbivorous)  urine  its  presence  is  usually  due 
to  a  fermentative  decomposition  of  hippuric  acid  whereby  benzoic 
acid  and  glycin  (glycocoU)  are  formed. 

CgHs  .  CO .  NH  .  CH2 .  COOH .  +  HoO 

=  CeHs .  COOH .  +  CH2  (NH2) .  COOH. 

'-  Cf.  Johnston  and  Church,  Chem.  of  common  life,  1880,  p.  147. 

2  Voit,  Unters.  ub.  d.  Einfl.  d.  Kochsalzes,  d.  Kaffees,  u.  s.  w.  Miinchen,  1860. 

"  Arch.  f.  Physiol.  Jahrg.  1886,  S.  72.  See  also  Kohert,  Arch.  f.  exp.  Path.  u. 
Pharm.  Bd.  xv.  '(1882),  S.  22,  and  cf.  Eossbach,  Pfliiger's  Arch.  Rd.  xxvii.  (1882), 
S.  372. 


186  HIPPUEIC   ACID. 

The  acid  is  usually  prepared  by  the  above  decomposition  of 
hippuric  acid,  which  is  readily  effected  by  a  short  boiling  with 
mineral  acids  or,  less  readily,  with  caustic  alkalis.  It  is  also 
obtained  by  the  dry  distillation  of  gum-benzoin  from  which  the 
acid  separates  by  sublimation.  The  sublimed  acid  generally 
crystallises  in  fine  needles  which  are  light  and  glistening.  It  is 
soluble  in  about  200  parts  of  cold  or  25  of  boiling  water  and  very 
soluble  in  alcohol,  ether,  and  petroleum-ether,^  in  which  latter 
hippuric  acid  is  insoluble.  When  precipitated  from  solutions,  either 
by  cooling  or  the  addition  of  acids  to  its  salts  in  the  cold,  the 
crystalline  form  is  usually  much  less  distinct. 

Apart  from  the  crystalline  form  benzoic  acid  is  characterised  by 
its  property  of  readily  subliming,  even  at  100°,  thus  resembling 
leucin  and  differing  markedly  from  hippuric  acid.  As  a  result  of 
this  it  passes  off  freely  in  the  vapours  arising  from  its  boiling 
aqueous  solutions,  so  that  in  concentrating  fluids,  such  as  urine,  in 
which  its  presence  is  conjectured,  they  should  be  first  rendered 
alkaline  with  sodium  carbonate,  thus  forming  a  non-volatile  salt. 
Benzoic  acid  may  be  additionally  recognised  by  the  following  test : 
when  treated  with  a  little  boiling  nitric  acid  and  evaporated  to 
dryness,  the  residue  thus  obtained  yields,  on  further  heating,  an 
unmistakable  odour  of  nitrobenzol. 

When  introduced  into  the  body  benzoic  acid  is  readily  and 
largely  converted  into  hippuric  acid,  while  at  the  same  time  small 
quantities  of  succinic  acid  may  at  the  same  time  make  their 
appearance.  The  chief  interest  in  the  acid  centres  in  the  above 
relationship  to  hippuric  acid,  a  fact  discovered  by  Wohler  in  1824 
and  specially  interesting  as  being  the  first  known  instance  of  a 
well  defined  synthesis  effected  by  the  animal  body,  and  the  start- 
ing-point for  the  disproval  of  Liebig's  views  as  to  the  funda- 
mental difference  in  the  metabolic  processes  of  animal  and  plant 
tissues. 

2.     Hippuric   acid.     CyHeOg.     [GJI, .  CO .  NH .  CH^ .  COOH.] 

(Benzoyl-gly  cin. ) 

This  acid  is  found  in  considerable  quantities  (1-5 -2*5  p.c.)  in 
the  urine  of  herbivora,  and  also,  though  to  a  much  smaller  amount 
(0-1 -1-0  grm.  per  diem)  in  the  urine  of  man.  It  is  undoubtedly 
formed  in  the  body  by  the  union,  with  dehydration,  of  benzoic 
acid  and  glycin  (see  §  419.)  This  mode  of  its  formation  may  be 
readily  observed  out  of  the  body  by  heating  together  dry  benzoic 
acid  and  glycin  in  sealed  tubes  to  160°. 

CeH5.COOH+CH2(NH2).COOH=C6H5.CO .  NH.CH^.COOH-f-H.O. 

1  Petroleum-ether  consists  ordinarily  of  a  mixture  of  the  more  volatile  hj-dro- 
carbons  obtained  by  distillation  during  the  fractionating  of  crude  petroleum,  and 
boils  up  to  about  120°.     The  most  volatile  petroleum-ether  boils  up  to  about  80°. 


CHEMICAL  BASIS  OF  THE  AKIMAL  BODY. 


187 


Its  constitution  is  further  cliaracteristically  shown  by  its  pro- 
duction by  the  action  of  benzamide  on  monochlor-acetic  acid :  — 

CeHs.CO .  NH2+CH2CI .  COOH=C6H5 .  CO.NH.CH2.  COOH.+HCl. 

and  also  by  that  of  benzoyl-chloride  on  glycin  :  ^  — 

CeHs-CO-Cl+CH,  (NH2) .  COOH^CeHs .  CO.NH.CH2.COOH+HCI. 

It  may  be  readily  obtained  from  the  urine  of  horses  or  cows, 
more  particularly  when  they  are  out  to  grass,^  the  perfectly  fresh  ^ 
urine  boiled  with  milk  of  lime  in  slight  excess,  by  which  means 
the  acid  is  fixed  as  a  hippurate  of  calcium.  It  is  then  filtered,  the 
filtrate  concentrated  to  a  small  bulk  and  treated  when  cold  with 
hydrochloric  acid  in  slight  excess :  this  decomposes  the  calcium 
salt,  liberating  hippuric  acid,  which  separates  out  at  once,  owing 
to  its  comparatively  slight  solubility.  It  is  then  purified  by  several 
recrystallisations  from  boiling  water,  but  it  is  extremely  difficult 
to  obtain  it  colourless. 


Fig.  31.     Hippuric  acid  crystals.     (After  Funke.) 

When  rapidly  separated  out  from  its  aqueous  solutions,  as  in 
the  above  method  of  its  preparation,  it  assumes  the  form  of  fine 
needles.  By  slower  crystallisation  it  yields  long  foursided  prisms 
or  columns  with  pyramidal  ends  ; .  these  are  frequently  arranged 
in  groups  and  present  a  semitransparent,  milky  appearance. 

When  pure  they  are  odourless  and  of  a  somewhat  bitter  taste. 
They  require  600  parts  of  water  for  their  solution  at  0°,  are  very 
readily  soluble  in  hot  water,  also  in  alcohol  and  to  a  less  extent  in 
ether.  They  are  conveniently  insoluble  in  petroleum-ether,  in 
virtue  of  which  hippuric  acid  can  be  readily  separated  from  benzoic 
acid  which  is  soluble  in  this  reagent.  Its  solutions  redden  litmus- 
paper. 

1  Baum,  Zt.f.  physiol  Chem.  Bd.  ix.  (1885),  S.  465. 

2  To  avoid  fermentative  decomposition  into  benzoic  acid  and  glycin. 


188  HIPPUEIC   ACID. 

Hippuric  acid  is  monobasic,  and  forms  salts  which  (except  the 
iron  salts)  are  readily  soluble  in  water:  from  these  solutions,  if 
sufficiently  concentrated,  excess  of  hydrochloric  acid  precipitates 
the  acid  in  fine  needles.  When  heated  with  concentrated  mineral 
acids  it  is  resolved  into  benzoic  acid  and  glycin.  The  same  de- 
composition occurs  readily  in  presence  of  putrefactive  organisms. 

Apart  from  the  characteristics  already  stated  the  acid  may  be 
recognised  by  the  following  reactions.  When  gently  heated  in  a 
small  tube  the  acid  does  not  at  once  sublime  as  does  benzoic  acid, 
but  melts  and  solidifies  again  on  cooling.  If  more  strongly  heated 
it  melts  as  before,  but  is  now  decomposed,  yielding  a  sublimate  of 
benzoic  acid  accompanied  by  an  odour  like  that  of  new  hay,  while 
oily  red  drops  are  observed  in  the  tube.  When  treated  with  boil- 
ing nitric  acid  (see  above  suh  benzoic  acid)  and  evaporated  to 
dryness  the  residue  on  being  heated  yields  the  marked  and 
characteristic  odour  of  nitrobenzol  (Liicke's  reaction  ^}.  As  al- 
ready stated  hippuric  acid  owes  its  formation  in  the  body  to  a 
union  of  benzoic  acid  with  glycin,  so  that  its  source  must  be 
sought  for  in  the  modes  by  which  benzoic  acid  (aromatic  sub- 
stance) is  introduced  into  or  arises  in  the  body.  The  source  is 
probably  of  more  than  one  kind.  Hay  and  grass  were  long  since 
stated  ^  to  contain  some  substance  which  yields  hippuric  acid  in 
the  body :  this  may  be  extracted  by  means  of  dilute  sulphuric 
acid,  less  readily  by  caustic  potash.'"^  More  recent  researches  have 
shown  the  presence  in  grass,  hay,  and  many  fruits  and  berries  not 
only  of  some  benzoic  acid  but  also  of  substances  such  as  quinic 
acid  (0H)4  CeHy .  COOH,  which  readily  yield  benzoic  acid  and  are 
hence  a  source  of  hippuric  acid.*  A  further  source  is  found  in 
the  aromatic  (benzoic)  products  of  the  putrefaction  of  proteids, 
such  as  in  especial  phenyl-propionic  acid  (CeHs .  CHj .  CHg .  COOH)^ 
which  in  its  amidated  form  is  more  particularly  a  product  of  the 
decomposition  of  vegetable  proteids,^  and  yields  benzoic  acid  by 
oxidation.  This  substance  has  been  found  in  the  rumen  of  cows 
fed  with  hay.''  These  facts  coupled  with  the  marked  occurrence 
of  putrefactive  changes  in  the  alimentary  canal  of  herbivora 
probably  account  for  the  preponderance  of  hippuric  acid  in  their 
urine.  In  carnivora  it  appears  that  some  traces  of  hippuric  acid 
may  be  observed  during  starvation,  originating  here  from  the 
aromatic  residues  of  the  tissue  proteids;  also  during  an  exclu- 
sively   meat-diet.^     When    fed    on   a    mixed   diet   some    of    the 

1  Arch.  f.  path.  Anat.  Bd.  xix.  (1860),  S.  196. 

2  Meissner  u.  Shepard,  Die  Hippiirfaure.     1866. 

3  Weiske,  Zt.f.  Biol.  Bd.  xii.  (1876),  S.  241. 

*  For  refs.  see  Salkowski  u.  Leube.     Die  Lehre  vom  Ham,  1882,  S.  131. 

5  E.  u.  H.  Salkowski,  Zuf.  phijsiol.  Chem.  Bd.  vii.  (1885),  S.  161. 

*>  Schulze  u.  Barbieri,  Ber.  d'.  d.  chem.  Ges.  1883,  S.  1711.  Jn.  f.  praJct.  Chem. 
(N.F.)  Bd.  XXVII.   (1883),  S.  337. 

'  Tappeiner,  Zt.f.  Biol.  Bd.  xxii.  (1886),  S.  236. 

^  Salkowski,  B./Ber.  d.  d.  chem.  Gesell.  1878,  S.  500.  Arch.  f.  path.  Anat.  Bd. 
73   (1878),   S,  421. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        189 

hippuric  acid  arises  from  the  benzoic  and  allied  constituents  of 
the  vegetable  part  of  the  food,  and  probably  not  an  inconsiderable 
amount  from  the  putrefactive  products  of  the  proteids  in  the  ali- 
mentary canal ;  in  accordance  with  this  it  is  found  that  disinfec- 
tion of  the  alimentary  canal  in  dogs  with  calomel  diminishes  the 
output  of  the  acid.i  Tyrosin,  notwithstanding  its  aromatic  con- 
stitution, does  not  give  rise  to  hippuric  acid  when  administered  to 
man. 2 

The  classical  researches  of  Bunge  and  Schmiedeberg  ^  have 
shown  that  the  synthetic  production  of  hippuric  acid  by  the 
union  of  benzoic  acid  and  glycin  takes  place  chiefly  in  the  kid- 
ney of  carnivora  (dogs).  In  herbivora  (rabbits)  it  appears  that 
a  considerable  formation  of  hippuric  acid  may  be  observed  on  the 
ingestion  of  benzoic  acid  even  after  exclusion  of  the  kidneys,*  and 
the  same  is  the  case  with  frogs.  Pathological  observations  on 
man  seem  to  indicate  that  in  them  the  kidneys  play  at  least  some 
part  in  the  synthetic  production  of  hippuric  acid  from  benzoic.^ 
When  benzoic  acid  is  administered  to  birds  it  reappears  in  the  ex- 
creta as  ornithuric  acid :  the  latter  when  boiled  with  hydrochloric 
acid  splits  up  into  benzoic  acid  and  ornithin,  the  latter  having  the 
composition  of  diamido-valerianic  acid.*^ 

3.  Tyrosin.  C9H11NO3 .  [OH .  CgH^ .  CH2 .  CH  .  (NK^)  .  COOH]. 
Para-oxyphenyl-a-amidopropionic  acid. 

The  earlier  work  on  the  synthesis  of  tyrosin  indicated  the  prob- 
able presence  in  its  molecule  of  some  aromatic  (phenyl)  radicle. 
The  more  recent  successful  synthesis  by  the  action  of  nitrous  acid 
on  para-amidophenyl  alanin  ''  has  confirmed  this  view  and  defi- 
nitely established  its  constitution.^  It  always  accompanies  leucin, 
though  less  in  amount,  as  a  product  of  the  pancreatic  digestion  of 
proteids,  but  not  of  gelatin,  also  as  a  product  of  their  putrefactive 
decomposition  as  well  as  of  the  action  of  boiling  mineral  acids  and 
alkalis.  It  is  also  perhaps  found  normally  in  small  quantities  in 
the  pancreas  and  its  secretion  and  in  the  spleen,  and  traces  have 
been  described  as  obtained  from  various  tissues  of  the  body.^  It 
is  normally  absent  in  urine,  but  makes  its  appearance  together  with 
leucin  in  this  excretion  in  several  diseased  conditions  of  the  liver, 
notably  acute  yellow  atrophy,  also  in  phosphorus  poisoning ;  there 

1  Baumann,  Zt.f.  physiol.  Chetn.  Bd.  x.  (1886),  S.  123. 

2  Baas,  Ibid.  Bd.  xi.'(1887),  S.  485. 

3  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  vi.  (1876),  S.  233.  Cf.  Schmiedeberg,  Ibid. 
Bd.  XIV.  (1881),  Sn.  288,  379.     See  also  Hoffmann,  A.  Ibid.  Bd.  vii.  (1877),  S.  233. 

*  W.  Salomon,  Zt.  f.  physiol.  Chem.  Bd.  in.  (1879),  S.  365. 

5  Jaavsveld  u.  Stolivis,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  x.  (1879),  S.  268. 

6  Jaffa',  Ber.  d.  d.  chem.  Gesell.  1877,  S.  1925  ;   1878,  S.  406. 

■^  Alanin  is  a-amidopropionic  acid ;  CHg  .  CH  (NHg) .  COOH. 
s  Erleumever  u.  Lipp.,  Ber.  d.  d.  chem.  Gesell.  1882,  S.  1544.     Liebig's  Annal. 
Bd.  219  (1883),  S.  161. 

3  V.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem.  Bd.  iv.  1878,  pp.  225,  227. 


190 


TYROSm. 


is  however  some  conflict  of  opinion  as  to  its  constancy  in  such  cases. 
It  is  also  present  in  not  inconsiderable  quantities,  along  with  leucin, 
in  many  plant  tissues. 

Tyrosin  crystallises  in  exceedingly  fine  needles  which  are  usu- 
ally collected  into  feathery  masses.  •  The  crystals  are  snow- 
white,  tasteless,  and  odourless.  If  crystallised  from  an  alkaline 
solution  tyrosin  often  assumes  the  form  of  rosettes  composed  of 
fine  needles  arranged  radiately. 

The  crystals  are  very  sparingly  soluble  in  cold  water  (1  in 
2000  at  20°),  much  more  soluble  in  boiling  water  (1  in  150)  ; 


Fig.  32.    Ttrosin  cktstals.     (Krukenberg.) 


they  are  almost  insoluble  in  strong  alcohol  (1  in  13500)  and 
quite  insoluble  in  ether.  They  are  readily  soluble  in  acids  and 
particularly  so  in  ammonia  and  other  alkalis  and  in  solutions  of 
alkaline  salts. 

Preparation,  (i)  The  products  of  a  prolonged  pancreatic  diges- 
tion of  proteids  are  neutralised  and  filtered ;  the  filtrate  when 
concentrated  usually  yields  crusts  of  tyrosin  crystals,  which  may 
be  readily  purified  by  solution  in  a  little  boiling  water  from 
which  they  separate  out  on  cooling  after  concentration  if  neces- 
sary, (ii)  Horn  shavings  are  boiled  for  24  hours  with  sulphuric 
acid  (5  of  acid  to  13  of  water).  The  sulphuric  acid  is  then  sepa- 
rated by  the  addition  of  lime,  and  the  filtrate  from  the  calcium 
sulphate  yields  as  before  crusts  of  tyrosin  crystals  on  concentra- 
tion and  cooling.  These  are  then  purified  by  recrystallisation 
from  boiling  water.i     Any  leucin  at  first  present  in  the  crystal- 

1  These  methods  suffice  for  the  preparation  of  small  amounts  of  tyrosin  for 
purposes  of  study.  For  full  details  of  its  preparation  and  most  productive 
separation  from  leucin  see  Hlasiwetz  and  Habermann,  quoted  sub  leucin.  See 
also  E.  Schulze,  Zt.  f.  physiol.  Chem.  Bd.  ix.  1885,  Sn.  63,  253,  on  the  separation 
of  amido-acids. 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.        191 

line  crusts  remains  in  the  mother-liquors  from  which  the  tyrosin 
has  been  separated. 

Apart  from  its  crystalline  form  and  characteristic  solubilities 
tyrosin  may  be  readily  recognised  by  several  well-marked 
reactions. 

Hoffmann's  reaction.  When  heated  with  Millon's  reagent  so- 
lutions of  tyrosin  yield  a  brilliant  crimson  or  pink  colouration 
which,  if  much  tyrosin  is  present,  is  accompanied  finally  by  a 
similarly  coloured  precipitate.  The  test  in  its  original  form  was 
applied  by  heating  with  a  solution  of  mercuric  nitrate  in  presence 
of  nitrous  acid.^ 

Firia's  reaction?  If  tyrosin  is  moistened  on  a  watch-glass  with 
concentrated  sulphuric  acid  and  warmed  for  five  or  ten  minutes 
on  a  water  bath,  it  turns  pink,  owing  to  the  formation  of  tyrosin- 
sulphonic  acid  —  C9H10  (SOoOH)  NO3  -f  2H2O.  This  is  then  diluted 
with  water,  warmed,  neutralised  with  barium  carbonate,  and  filtered 
while  hot.  The  filtrate  yields  a  violet  colour  on  the  careful  addi- 
tion of  very  dilute  perchloride  of  iron.  The  colour  is  readily  de- 
stroyed by  any  excess  of  the  iron  salt.^ 

The  remarks  made  on  p.  149  on  the  optical  properties  of  leucin, 
apply  also  to  tyrosin.* 

When  tyrosin  is  subjected  to  putrefactive  decomposition  it  yields 
paraoxyphenylacetic  acid  OH.  C6H4-CH2.  COOH.,  paraoxyphenyl- 
propionic  (hydroparacumaric)  acid  OH  .  C6H4  -  CH2  .  CH, .  COOH., 
/8-phenylpropionic-(hydrocinnamic)  acid  CeHs  .  CHg  .  CH2  .  COOH., 
phenol,  CgHs  .  OH.,  and  parakresol  CHg  .  C6H4  .  OH.^  These  sub- 
stances occur  normally  in  small  and  variable  amounts  in  urine  and 
are  increased  in  quantity  in  this  excretion  by  the  administration  of 
tyrosin.  Their  presence  is  without  doubt  chiefly  due  to  putrefactive 
processes  occurring  in  the  alimentary  canal  in  correspondence  with  the 
facts  that  the  bodies  in  question  are  found  most  markedly  in  the  urine 
of  herbivora,  in  increased  quantity  in  that  of  men  under  a  vegetable 
diet,  and  largely  disappear  under  the  influence  of  drugs  such  as  calo- 
mel, which  lessens  or  prevents  the  occurrence  of  putrefactive  changes 
in  the  intestine.^  In  the  absence  of  these  putrefactive  processes  ty- 
rosin when  administered  m  not  excessive  amounts  is  apparently  com- 
pletely oxidised  and  does  not,  as  frequently  stated,  give  rise  to  any 
increased  output  of  urea.'''     In  large  doses  tyrosin  reappears  externally 

1  Liebig's  An7ial.  Bd.  lxxxvii.  (1853),  S.  124. 

2  Liebig's  Ajinal.  Bd.  lxxxii.  (1852),  S.  231. 

3  For  other  less  important  reactions  see  Wurster,  Centralh.  f.  Physiol.  Bd.  i. 
(1887),  S.   194.     Udranszky,  Zt.  f.  phjsiol.  Chem.  Bd.  xii.  (1888),' S.  355. 

*  For  details  see  Mauthner,  Monatsb.  f,  Chem.  Bd.  ill.  (1882),  also  Sitzb.  d. 
Wien.  Akad.  Bd.  lxxxv.  (1882),  April-Hft.  Schulze,  Zt.  f.  phi/siol.  Chem.  Bd.  ix. 
(1885),  Sn.  98,  109.     Lippmann,  Ber.  d.  d.  chem.  Gesell.  1884,  S.  2838. 

5  Weyl,  Zt.  f.  physiol.  Chem.  Bd.  iii.  (1879),  S.  312.  Baumann,  Ibid.  Bd.  iv. 
S.  304.  Schotten,  Ibid.  Bd.  vii.  (1882),  S.  23.  Salkowski,  E.  u.  H.  Ibid.  S.  450. 
Baumann,  Ibid.  S.  553. 

6  Baumann,  Zt.  f.  physiol.  Chem.  Bd.  x.  (1886),  S.  129. 

''  Schultzen  u.  JSTencki,  Zt.  f.  Biol.  Bd.  viii.  (1872),  S.  124.  Kiissner,  Inauc;. 
Diss.  Konigsberg,  1874.  Brieger,  Zt.  f.  physiol.  Chem.  Bd  ii.  (1878),  S.  241, 
Rohmann,  Berl.  Iclin.  Wochensch.  1888.  Nrn.  43,  44.  Cohn,  Zt.  f.  physiol.  Chem 
Bd.  XIV.  (1819),  S.  200. 


192 


TYROSIN. 


NH.  CO 

in  the  form  of  tyrosin-hydantoin  ^  OH  .  CgHi  -  C2H3  ^  | 

^CO.     NH 
This  substance  is  the  anhydride  of  tyrosin  hydantoic  acid  ^ 

OH  .  C6H4  -  C2H3  (NH  .  CO  .  NH2)  COOH. 

and  analogous  to  the  similar  compounds  excreted  after  the  ingestion 
of  sarkosin  and  taurin.  (See  pp.  141,  143.)  It  yields  tyrosin,  am- 
monia, and  carbonic  dioxide  when  heated  with  baryta  in  sealed 
tubes. 

4.    Kynurenic  acid.    C10H7NO3.   [CaHsN  .  OH .  COOH.]     Oxy- 

chinolin-carboxylic  acid. 

This  acid  occurs  characteristically  but  in  variable  amounts  in 
the  urine  of  dogs,  but  does  not  appear  to  have  been  found  normally 
in  that  of  man.  It  was  first  described  by  Liebig.^  It  is  most 
readily  separated  horn,  fresh  urine  by  precipitation  with  phospho- 
tungstic  acid  after  the  addition  of  hydrochloric  acid ;  it  is  then 


Fig.  33.     Crystals  of  Kynukenic  acid.     (After  Kiihne.) 

liberated  from  the  precipitate  by  the  action  of  baryta.*  It  may 
also  be  obtained  by  concentrating  the  urine  to  one-third  of  its 
bulk,  acidulating  with  hydrochloric  acid  and  allowing  it  to  stand 
in  a  cool  place  for  several  days  until  the  separation  of  the  acid  is 
complete.^  It  may  be  separated  from  admixed  uric  acid  by  solu- 
tion in  dilute  ammonia.  It  is  practically  insoluble  in  cold  water, 
slightly  so  in  boiling  water,  and  readily  soluble  in  hot  alcohol  and 

1  Blender mann,  Ibid.  Bd.  vi.  (1882),  S.  234. 

2  JafEe,  Ibid.  Bd.  vii.  (1883),  S.  306. 

3  Liebig's  Annalen,  Bd.  86  (1853),  S.  125,  Bd.  108  (1858),  S.  354. 

*  Hofmeister,   Zt.   f.  physiol.   Ckem.  Bd.  v.    (1881),  S.   67.      Cf.  Briefer,   Ibid. 
Bd.  IV.  S.  89.  ^ 

^  Schmiedeberg  u.  Schultzen,  Liebig's  Annalen,  Bd.  clxiv.  (1872),  S.  155. 


CHEMICAL  BASIS   OF   THE  ANIMAL   BODY..      193 

in  dilute  ammonia.  It  crystallises  in  long  brilliant  white  needles 
which  when  kept  under  acidulated  water  are  often  changed  into 
long  glittering  foursided  prisms. 

This  acid  forms  salts  of  which  that  with  barium  crystallises 
readily  and  in  a  very  characteristic  triangular  form. 

Apart  from  its  crystalline  form  and  that  of  its  barium  salt  this 
acid  may  be  readily  recognised  by  the  following  reaction.  When 
heated  on  a  water  bath  with  hydrochloric  acid  and  chlorate  of 
potash  and  evaporated  to  dryness  a  reddish  residue  is  obtained, 
which  turns  at  first  to  a  brownish  green  on  the  addition  of  am- 
monia, and  finally  to  an  emerald  green.^ 


Fig.  34.     Crystals  of  bakium  Kynurenate.     (After  Kiihne.) 

By  prolonged  heating  to  250  —  260°  kynurenic  acid  evolves 
carbonic  anhydride  and  is  converted  into  kynurin  (oxychinolin) 
CgHelSr  (OH),  and  when  heated  with  zinc  dust  in  a  current  of 
hydrogen  it  is  converted  into  chinolin  CgHelSr  (OH)  -|-  H2  = 
C9H7N  -\-  H2O.  These  reactions  throw  considerable  light  on  the 
constitution  of  the  acid.^ 

The  amount  of  kynurenic  acid  in  the  urine  is  increased  on  the 
ingestion  of  isatin,  a  product  of  the  oxidation  of  indigo.^  Under 
ordinary  conditions  its  amount  in  this  excretion  is  dependent  upon 
the  nature  of  the  food  supplied  to  the  animal,  being  greatest  under 
a  proteid  diet,  and  is  not  related  to  the  occurrence  or  absence  of 
putrefactive  processes  in  the  alimentary  canal.* 

5.   Phenol.    CeHj .  OH.  Oxybenzol.    (Carbolic  or  phenyhc  acid.) 

This  substance  is  formed,  together  with  indol  and  skatol,  dur- 
ing the  putrefactive  decomposition  of  proteids,  more  especially  in 
prolonged  putrefactive  pancreatic  digestions.^     From  these  it  may 

1  Jaffe,   Zt.  f.  physiol.  Chem.  Bd.  vii.  (1882-3),  S.  399. 

2  Kretschy,  Ber.  d.  d.  chem.  Gesell.  Bd.  xii.  (1879),  S.  1673.  Monatsh.  f.  Chem 
Bd.  II.  (1881),  S.  57. 

^  Niggeler,  Arch.f.  exp.  Path.  u.  Pharm.  Bd.  iii.  (1874),  S.  67. 

*  Baumann,  Zt.  f.  physiol.  Chem.  Bd.  x.  (1886),  S.  131.  But  cf.  Haagen,  Inaug.- 
Diss.,  Konigsb.,  1887.     (See  Centralb.  f.  d.  Med.  Wiss.  1889,  S.  214.) 

5  Baumann,  Zt.  f.  physiol.  Chem.  Bde.  i.  (1877),  S.  60,  in.  250.  Brieger,  Ibid. 
Bd.  III.  (1879),  S.  134.     Odermatt,  Jn.f.  prakt.  Chem.  Bd.  xviii.  (1878),  S.  249. 

13 


194  PHENOL. 

be  obtained  by  simple  distillation.  In  accordance  with  this  it  is 
formed  in  not  inconsiderable  quantity  in  the  alimentary  canal, 
more  especially  when  putrefactive  processes  in  its  contents  are 
increased  either  pathologically  or  as  the  result  of  experimental 
interference.^  On  the  phenol  thus  formed  a  small  proportion  is 
passed  out  in  the  faeces,''^  the  larger  part  however  is  excreted  in 
the  urine  as  an  ethereal  salt  of  sulphuric  acid,  viz.  phenylsul- 
phate  of  potassium.  The  latter  is  typical  of  an  extensive  series 
of  similar  ethereal  sulphates  which  make  their  appearance  in 
urine  after  the  ingestion  of  aromatic  substances. 

Their  nature  and  constitution  was  first  definitely  ascertained  by 
Baumann,^  although  it  had  previously  been  shown  that  phenol, 
even  after  it  has  been  administered  as  such,  does  not  exist  in  the 
free  state  in  urine  but  may  be  set  free  by  distillation  with  a 
mineral  acid.^ 

Fhenyl-sulphuric  acid.^  CeHs .  0  .  SO2OH.  Apart  from  its 
abundant  presence  in  urine  as  an  alkaline  salt  after  the  admin- 
istration of  phenol  this  compound  occurs  normally  in  small 
quantities  in  most  urines,  more  particularly  in  those  of  herbivora, 
since  in  these  animals  the  conditions  for  its  formation  are  espe- 
cially provided  by  the  preponderance  of  aromatic  compounds  in 
their  food  and  the  more  marked  activity  of  putrefactive  changes 
in  their  alimentary  canal.  The  total  sulphates  in  urine  consist 
therefore  partly  of  this  ethereal  sulphate  (together  with  the 
similar  compounds  of  kresol,  indol,  and  skatol,  see  helow)  and  of 
ordinary  sulphates.  The  relative  amounts  of  the  sulphuric  acid 
contained  in  these  two  forms  is  ascertained  by  acidulating  with 
acetic  acid  and  adding  barium  chloride,  by  which  the  sulphuric 
acid  present  as  ordinary  sulphates  is  precipitated  as  barium  sul- 
phate. The  filtrate  from  this  is  now  boiled  with  hydrochloric 
acid,  by  whose  action  the  ethereal  sulphates  are  decomposed, 
yielding  phenol  and  sulphuric  acid,  which  again  forms  barium 
sulphate ;  from  this  the  amount  of  the  ethereal  salts  of  sulphuric 
acid  may  be  at  once  determined.^     While  the  probable  mode  of 

1  E.  Saikowski,  Ber.  d.  d.  chem.  Gesell.  1876,  S.  1595.  Ibid.  1877,  S.  842.  Cen- 
tralb.  f.  d.  Med.  Wiss.  1876,  S.  81g.  Arch.  f.  Phiislol.  Jahrg.  1877,  S.  476.  Brieger, 
Zt.  f.^phiisiol.  Chem.  Bd.  ii.  (1878),  S.  241.    G.  Hoppe-Seyler,  Ibid,  Bd.  xn.  (1888),  S.  1. 

2  Brieger,  Ber.  d.  d.  chem.  Gesell.  1877,  S.  1027.  Jn.  f.  prakt.  Chem.  Bd.  xvii. 
(1878),  S.  134. 

3  Pfliiger's  Arch.  Bd,  xiii.  (1876),  S.  285.  Ber.  d.  d.  chem.  Gesell.  1876,  S.  55. 
Baumann  und  Herter,  Zt.  f.  physlol.  Chem.  Bd.  i-  (1877),  S.  244.  See  also 
Baumann,  Ibid.  Bd.  ii.  (1878),  S.  335,  Bd.  x,  (1886),  S.  123.  For  a  list  of 
substances  which  when  administered  leave  the  body  as  ethereal  sulphates,  see 
Hermann's  Hdbch.  d.   Physiol.   Bd.   v.  Th.    1,  S.  508. 

*  Buliginsky,  Hoppe-Seyler's  Med.  chem.  Unters.  Hft.  2,  1866,  S.  234.  Hoppe- 
Seyler,  Pfluger's  Arch.  Bd.  v.  (1872),  S.  470. 

^  Not  to  be  confounded  with  phenolsulphonic  acid,  C6H4  (OH) .  SO2  OH. 

6  For  the  accurate  separation  of  the  ethereal  sulphates  which  usually  occur 
mixed  in  urine,  some  special  works  should  be  consulted,  such  as  Neubauer  u.  Vogel, 
Analyse  des  Harris,  or  Saikowski  u.  Leube,  Die  Lehre  vom  Ham.  Cf.  Baumann,  Zt 
f.physiol.  Chem.  Bd.  i.  (1876),  S.  70,  Ibid.  Bd.  vi.  (1882),  S.  183. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        195 

formation  of  this  acid  is  undoubtedly  due  to  the  primary  produc- 
tion of  phenol  by  putrefactive  processes  from  proteids^  and  the 
subsequent  colligation  of  this  phenol  with  sulphuric  acid,  very 
little  is  known  of  the  seat  or  mode  of  this  union.  It  has  not 
been  definitely  connected^  if  at  all,  with  any  distinctly  synthetic 
activity  of  the  kidney.  ^ 

Since,  as  has  been  said,  phenol  does  not  exist  in  the  free  state 
in  urine,  its  detection  necessitates  the  decomposition  of  its  com- 
pound, viz.  the  phenylsulphate  of  potassium.  This  is  best  brought 
about  by  distilling  the  urine  (200  c.c.)  with  strong  hydrochloric 
acid  (40  c.c.)  or  5  p.c.  of  sulphuric  acid  until  about  150  c.c.  of 
distillate  has  passed  over.  The  distillate  contains  free  phenol, 
which  is  tested  for  qualitatively  by  the  reactions  described  below, 
and  estimated  quantitatively  by  the  formation  of  a  compound 
with  bromine,  tribromphenol,  C6H2Br3 .  GH.^ 

Phenol  reactions  (i).  A  violet-blue  colouration  on  the  addition 
of  neutral  solutions  of  perchloride  of  iron.  This  colour  is  similar 
to  that  yielded  by  salicylic  acid,  but  the  absorption  spectra  of  the 
two  are  stated  to  be  different.^  It  is  destroyed  by  excess  of  the 
reagent  and  is  also  not  obtained  in  presence  of  acids  and  alkalis 
or  of  alcohol.*  (ii)  When  a  solution  of  phenol  is  mixed  with 
one  quarter  of  its  bulk  of  ammonia  and  a  few  drops  of  chloride 
of  lime  solution  (1  to  20  of  water)  and  gently  warmed  it  yields  a 
blue  colouration.^  (iii)  When  boiled  with  Millon's  reagent  a 
marked  and  persistent  pink  or  red  colour  similar  to  that  yielded 
by  ty rosin  is  obtained.^  (iv)  Mere  traces  of  phenol  give  a  yel- 
lowish crystalliae  precipitate  on  the  addition  of  bromine  water. 
This  reaction  is  used  as  stated  above  for  the  quantitative  estima- 
tion of  phenol.  Of  these  reactions  (iii)  and  (iv)  are  the  most 
delicate,  (v)  On  the  addition  of  furfurol  (C5H4O2,  aldehyde  of 
pyromucic  acid)  solution  (-5  p.c.)  and  strong  sulphuric  acid,  phenol 
yields  a  brilliant  red  colour  which  finally  turns  to  blue.^ 

6.     Kresol.     C6H4.OH.CH3.     Methylphenol. 

This  homologue  of  phenol  exists  in  three  isomeric  forms,  orthb-, 
para-,  and  metakresol.  It  is  now  known  that  the  phenols  which 
may  be  obtained  by  the  distillation  of  urine  with  acids  consist 
preponderatingly  of  parakresol,  accompanied  in  some  cases  by 
orthokresol  and  possibly  (?)  by  metakresol  in  minute  amounts. 
Like  phenol  it  is  not  found  free  in  urine,  but  as  kresylsulphuric 

1  Christiani  u.  Baumann,  Zt.  f.  physiol.  Chem.  Bd.  ii.  (1878),  S.  350,  See  also 
Kochs,  Pfliiger's  Arch.  Bd.  xx.  (1879),  S.  64. 

^  Landoit,  Ber,  d.  d.  chem.  Gesell.  1871,  S.  770. 

^  Krukenberg,  VerhandL  d.  physilc.-med,  Gesell.  zu  Wilrzhurq,  Bd.  xvili.  (1884), 
S.  197. 

*  Hesse,  Liebig's  Annal.  Bd.  182  (1876),  S.  161. 

5  E.  Salkowski,  Pfliiger's  Arch.  Bd,  v.  (1872),  S.  353. 

6  Plugge,  Zt.  f.  anal.  Chem.  Bd.  xi.  (1872),  S.  173  See  also  Alme'n,  Ibid.,  Bd. 
XVII.  (1878),  S.  107. 

7  Udranszky,  Zt.f.  physiol.  Chem.  Bd.  xii.  (1888),  Sn.  355,  377. 


196  PYEOCATECHIN, 

acid,^  C7H7O  .  SO2OH.  The  general  conditions  of  its  presence  in 
urine  are  practically  identical  with  those  for  the  occurrence  of 
phenylsulphuric  acid.^  When  introduced  into  the  animal  body 
the  three  isomeric  kresols  undergo  distinctly  different  oxidational 
changes."^ 

Eeactions.  On  the  addition  of  an  excess  of  bromine  water  to 
its  solutions  parakresol  yields  a  brominated-  derivative,  but  the 
compound  is  only  obtained  in  a  separate  and  crystalline  form 
after  prolonged  standing,  differing  characteristically  from  the 
analogous  compound  of  phenol,  which  under  similar  circumstances 
is  formed  rapidly.  It  yields  a  reddish  yellow  colouration  with 
potassium  nitroprusside  and  caustic  potash,  which  turns  bright 
pink  on  the  addition  of  an  excess  of  acetic  acid.*  Aceton  gives 
a  similar  reaction.  With  furfurol  and  sulphuric  acid  the  reaction 
is  closely  similar  to  that  which  phenol  gives.^ 

7.     Pyrocatechin.     Q^i  (0H)2.     Orthodioxybenzol. 

This  substance  occurs,  in  small  amounts  in  human  urine  united 
with  sulphuric  acid  as  a  mono-ethereal  compound  OH  .  C6H4 .  0  . 
SO2OH.  It  is  more  plentifully  present  in  the  urine  of  herbivora, 
especially  of  the  horse,  and  is  largely  increased  in  amount  by  the 
administration  of  benzol  or  phenol.^  It  is  also  stated  to  occur  in 
cerebrospinal  fluid."  When  present  in  urine  it  (together  with 
hydrochinon)  confers  on  this  excretion,  especially  if  alkaline,  the 
property  of  turning  successively  greenish,  brown,  and  finally  dark- 
brown  or  almost  black  on  exposure  to  the  air,  and  of  readily  re- 
ducing solutions  of  metallic  salts,  a  fact  to  be  taken  into  account 
when  dealing  with  the  presence  or  absence  of  sugar  in  the  urine. 
Solutions  of  pyrocatechin  turn  emerald  green  on  the  addition  of 
a  few  drops  of  very  dilute  solution  of  ferric  chloride,  avoiding  all 
excess  of  the  reagent.  If  the  green  solution  is  now  acidulated 
with  tartaric  acid,  it  turns  violet  on  the  subsequent  addition  of  a 
little  ammonia  and  purplish-red  on  the  addition  of  excess.  The 
green  colour  may  be  restored  by  excess  of  acetic  acid.^     It  may 

1  Baumann,  Ber.  d.  d.  chem.  Gesell.  Bd.  ix.  (1876),  S.  1389.  Zt.  f.  physiol. 
Chem.  Bd.  ii.  (1878),  S.  335.  Preusse;  Ibid.  S.  355.  Brieger,  Ibid.  Bd.  iv. 
(1880),   S.  204. 

2  Baumann  u.  Brieger;  Ibid.  Bd.  iii.  (1879),  S.  149.  Baumann,  Ibid.  iv.  S. 
304.  For  the  detection  and  separation  of  the  kresols  and  phenol  see  Baumann 
u.  Brieger,  Ber.  d.  d.  chem.  Gesell.  Bd.  xii.  (1879),  S.  804.  Baumann,  Zt.  f. 
physlol.  Chem.  Bd.  vi.  (1882),  S   183.     Brieger,  Ibid.  viii.  (1883),  S.  311. 

'3  Preusse,  Ibid.  Bd.  v.  (1881),  S.  57. 

*  V.  Jacksch,  Zt.f.  klin.  Med.  Bd.  viii.  (1884),  S.  130. 

5  Udranszky,  cit,  (sub  phenol). 

6  See  Baumann,  Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  63,  Baumann  u.  Herter,  Zt. 
f.  pliysioL  Chem.  Bd.  I.  (1877),  S.  248,  Baumann  u.  Preu.sse,  Ibid.  Bd.  iii.  (1879),  S. 
156.  Brieger,  Arch.  f.  phijsiol.  Jahrq.  1879,  Suppl.-Bd.  S.  61.  Nencki  u.  Giacosa, 
Zt.  f.  phi/siol.  Cliem.  Bd,  'iv.  (1880),'  S.  325.  Schmiedeberg,  Arch.  f.  exp.  Path.  u. 
Pharm.  Bd.  xiv.   (1881),  S.  288. 

■^  Halliburton, .//.  of  Phi/siol.  Vol.  x.  (1889),  p.  247. 

*  Ebstein  u.  Miiller,  Virchow's  Arch.  Bd.  lxv.  (1875),  S.  394.  See  also  Jacquemin, 
Rev.  Med.  de  I'Est.  T.  \iii.  (1817),  Y,.  90. 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.        197 

be  distinguished  from  hydrochinon  by  yielding  a  precipitate  with 
normal  acetate  of  lead  which  is  soluble  in  acetic  acid,  whereas  the 
latter  substance  does  not.  No  simple  directions  can  be  given  for 
the  separation  and  estimation  of  pyrocatechin  in  presence  of 
phenol,  kresol,  and  hydrochinon.^ 

But  little  is  known  as  to  the  source  of  this  substance  in  urine 
apart  from  its  probable  formation  from  the  phenol  produced  by 
putrefactive  changes  in  the  alimentary  canal.  In  herbivora  there 
is  some  evidence  that  it  is  derived  from  certain  aromatic  consti- 
tuents of  their  food.^ 

8.     Hydrochinon.    C6H4  (0H)2.     Paradioxybenzol. 

Has  not  yet  been  described  as  occurring  normally  in  urine,  but 
only  as  the  result  of  the  ingestion  of  phenol.  It  exists  in  urine 
as  an  ethereal  compound  with  sulphuric  acid,  and  is  largely  the 
cause  of  the  dark  colour  which  this  excretion  assumes  after  the 
absorption  of  phenol  on  exposure  to  the  air.  It  resembles  pyro- 
catechin in  effecting  the  reduction  of  metallic  salts,  but  differs 
from  it  in  being  nearly  insoluble  in  cold  benzol  and  in  not  yield- 
ing any  precipitate  with  normal  lead  acetate.  This  latter  property 
suffices  for  its  separation  from  pyrocatechin.  It  is  readily  con- 
verted by  oxidation  into  chinon  C6H4O2  whose  characteristic  odour 
affords  a  further  means  of  identification,  and  when  heated  in  an 
open  test-tube  it  yields  a  blue  sublimate.^ 

The  third  known  isomeric  dioxybenzol,  viz.  meta-dioxybenzol 
or  resorcin,  has  not  yet  been  found  in  the  animal  body  or  in 
urine. 

The  Indigo  Series. 


NH 
1.     Indol.     CgH.N.     I  CeH^;         )CH. 

CH-^ 


C6H4 


Indol  occurs  characteristically  in  the  fgeces,  to  which  with 
skatol  it  imparts  their  peculiarly  unpleasant  odour.*  Its  presence 
here  is  due  to  its  formation  during  the  putrefactive  decomposition 
of  proteids  which  usually  occurs  to  a  greater  or  less  extent  in  the 
alimentary  canal,  part  of  the  indol  leaving  the  body  in  the  urine 
as  a  potassium  salt  of  indoxylsulphuric  acid  {see  below'),  the 
remainder  being  excreted  with  the  fcsces.  It  may  readily  be 
obtained,   contaminated    by   varying   quantities    of    phenol   and 

1  See  Baumann,  Zt.  f.  physiol  Chem.  Bd.  vi.  (1882),  S.  183.  Schmiedeberg,  loc. 
cit.  S.  304. 

2  Preusse,  Zt.f.  physiol.  Chem.  Bd.  ii.  (1878),  Sn.  324,  329. 

3  In  addition  to  the  literature  preceding!}^  quoted,  see  more  particularly 
Baumann  u.  Preusse,  Arch.  f.  physiol.  Jahrg.  1879,  S.  245.  Brieger,  Ihid.  Suppe- 
Hft.  S.  66,  Baumann  u.  Preusse,  Zt.  f.  physiol.  Chem.  Bd.  vii.  (1889),  S.  156. 
Baumann,  Ibid.  Bd.  vi.  (1882),  S.  188. 

*  Eadziejewski,  Arch.  f.  Anat.  u.  Physiol.  1870,  S.  42. 


198  INDOL. 

skatol  (see  helow),  by  acidulating  and  distilling  the  products  of  a 
not  too  prolonged  alkaline  ^^uto^^fadive  pancreatic  digestion  of 
proteids,  preferably  of  liver  or  fibrin.  Indol  passes  over  into  the 
distillate,  from  which  it  is  extracted  by  shaking  up  with  ether, 
and  is  left  behind  as  an  impure  oily  liquid  when  the  ether  is 
driven  off  by  heat.^  It  may  also  be  prepared  by  heating  moist 
proteids  slowly  to  a  red-heat  with  excess  of  caustic  potash,  the 
indol  as  before  passing  over  into  the  distillate.^  Indol  is  a  crys- 
talline body  which  when  pure  melts  at  53°.  It  is  soluble  in 
boiling  water,  alcohol,  and  ether, 

'  Reactions.  A  strip  of  pine-wood  moistened  with  hydrochloric 
acid  is  coloured  bright  crimson  when  dipped  into  an  alcoholic 
solution  of  indol.3  Its  alcoholic  solution  turns  red  when  treated 
with  nitrous  (fuming  nitric)  acid,  and  its  aqueous  solution  gives  a 
copious  red  precipitate  with  the  same  reagent.*  This  reaction  is 
more  delicate  if  carried  on  by  the  addition  of  strong  nitric  acid 
first,  and  of  a  2  p.c.  solution  of  potassium  nitrite  subsequently.^ 
When  indol  in  dilute  solution  is  mixed  with  a  little  sodium  nitro- 
prusside  and  then  with  a  few  drops  of  caustic  soda  it  turns  at 
once  violet-blue,  and  pure  blue  on  subsequent  acidulation  with 
acetic  acid.^  Skatol  yields  neither  of  the  above  reactions.  Indol 
also  forms  a  well-marked  crystalline  compound  with  picric  acid 
(trinitro-phenol)  when  added  in  benzolic  solution  to  a  solution  of 
the  acid  in  benzol,  so  also  does  skatol. 

It  has  been  already  stated  that  a  part  of  the  indol  formed  in  the 
alimentary  canal  leaves  the  body  in  the  urine  as  a  potassium  salt  of 
indoxylsulphuric  acid;  by  oxidation  this  may  be  readily  decomposed 
into  indigo-blue  and  acid  potassium  sulphate: — 2C8H6NKSO4 -)- O.2 
=  Ci6Hioi^202 -t-SKHSO^.^  By  the  action  of  powerful  reducing 
agents  indigo-blue  may  be  made  to  yield  indol,  which  by  oxidation 
may  be  again  converted  into  indigo-blue.  This  shows  that  iiidol  is 
the  mother  substance  of  the  indigo  series.  The  constitution  of  indol 
is  elucidated  bj^  its  formation  from  orthonitrophenylchlorethylene 
C6H4  (NO2)  -  CH  =  CHCl.  When  this  is  reduced  with  tin  and  hydro- 
chloric acid  it  yields  CgH4  (NHo)  —  CH=:CHCb  and  this  when 
heated  to  160°— 170°  with  sodium-ethylate  (NaO .  C2H5)  yields 
sodium  chloride,  ethyl-alcohol  and  indol. ^ 

1  Neiicki,  Ber.  d.  d.  chem.  Gesell.  Bde,  vii.  (1874),  S.  1.593,  viii.  S.  336,  722. 
Brieger,  Zt.  f.  phi/sio!.  Chem.  Bd.  iii,  (1879).  S.  134.  Cf.  Koukol-Yasnopolsky, 
Pfliiger's  Arch.  Bd.  xii.  (1876),  S.  78.  Baumann,  Zt.  f.  phiisiol.  Chem.  Bd.  i. 
(1877),  S.  63.  Weyl,  Ibid.  S.  339.  See  specially  E.  Salkowski,  Ibid.  Bd.  viii. 
(1884),  S.  417. 

2  Kiihne,  Ber.  d.  d.  chem.  Gesell.  Bd  viii.  (1875),  S.  206.  Nencki,  Jn.  f.  prakt. 
Chem.  (N.  F.),  Bd,  xvii.  (1878).  S.  97. 

3  This  reaction  depends  on  the  presence  of  coniferin  in  the  pine-wood.  Phenol 
under  similar  conditions  yields  a  blue  colouration.  But  see  Udranszky,  Zt.  f. 
physiol  Chem.  Bd.  xii.  (1888),  S.  367. 

*  Cf.  Nencki,  Ber.  d.  d.  chem.  Gesell.  Bd.  viii.  (1875),  S.  722. 

6  E.  Salkowski,  loc.  cit. 

6  Legal,  Bresl.  drtzl.  Zeitsch.  Nrn.  3  u.  4,  1883. 

''  Baumann  u.  Brieger,  Zt.  f.  physiol.  Chem.  Bd.  in  (1879),  S   254. 

8  Lipp,  Ber.  d.  d.  chem.  Gesell.  Bd,  xvii.  (1884),  S.  1067. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.        199 

2.  Indoxylsulphuric  acid.  CgHgN  .  0  .  SOgOH.  The  indican 
of  urine 

A  substance  was  long  ago  described  as  frequently  occurring  in 
the  urine  and  sometimes  in  the  sweat  of  man  and  other  animals 
wliich  yielded  by  the  action  of  acids  the  blue  colouring  matter 
indigo  as  one  of  the  products  of  its  decomposition.  It  was  re- 
garded at  that  time  as  identical  with  the  indican  known  to  occur 
in  several  plants  (Indigofera  tinctoria,  Isatis  tinctoria).  Hoppe- 
Seyler  on  the  other  hand,  having  regard  to  the  greater  ease  with 
which  the  indican  of  plants  undergoes  decomposition,  regarded 
them  as  most  probably  different  substances.^  This  view  was  con- 
firmed by  the  researches  of  Baumann,  who  first  proved  that  urinary 
indican  is  not  a  glucoside,  as  is  that  of  plants,  but  is  in  reality 
an  ethereal  compound  of  sulphuric  acid  with  indoxyl  (CgHelsr .  OH) 
analogous  to  those  already  described  above  as  derived  from  phenol, 
kresol,  &c.^  Indol,  as  previously  stated,  is  a  characteristic  product 
of  the  putrefaction  of  proteids.  Further,  when  administered  to 
animals,  it  leads  to  a  correspondingly  increased  output  of  urinary 
indican,^  an  increase  which  is  similarly  observed  as  the  result  of 
either  a  normally,  pathologically,  or  experimentally  increased 
activity  of  putrefactive  processes  in  the  alimentary  canal.*  Hence 
indican  is  under  normal  conditions  more  plentiful  in  the  urine  of 
herbivora  than  of  carnivora.  It  is  also  increased  in  carnivorous 
urine  under  a  meat  diet,  is  not  increased  by  the  a*dministration  of 
gelatin  and  is  least  during  starvation,  although  in  the  latter  case 
it  may  not  entirely  disappear.^  These  facts  correspond  again  to 
the  experimental  observations  that  gelatin  does  not  yield  indol 
during  its  putrefactive  decomposition,^  whereas  mucin  does,'^  and 
the  latter  substance  constitutes  a  part  at  least  of  the  contents  of 
the  alimentary  canal  during  starvation.  These  statements  show 
clearly  the  origin  and  mode  of  formation  of  urinary  indican,  the 
first-formed  indol  undergoing  oxidation  into  indoxyl,  which  is 
subsequently  united  to  the  elements  of  sulphuric  acid  and  excreted 
as  an  ethereal  compound 

Indoxyl-sulphuric  acid  is  not  known  in  the  free  state ;  its  most 
important  salt  is  that  with  potassium,  the  form  in  which  it  occurs 

1  For  earlier  literature  see  Hoppe-Seyler's  Physiol. -path.  chem.  Anal.  Aufl.  4, 
1875,  S.  191  ;  and  Physiol.  Chem.  1881,  S.  841. 

2  Pfliiger's  Arch.  Bd.  xiii.  (1876),  S.  301  ;  Zt.  f.  physiol  Chem.  Bd.  i.  (1877), 
S.  60;  III.  (1879),  S.  254.  Cf.  G.  Hoppe-Seyler,  Ihiil.  Bde.  vii.  (1883),  S.  403;  vin. 
S.  79. 

3  Jaffe',  Centralb.  f.  d.  med.  Wiss.  1872,  Sn.  2,  481,  497.  Yirchow'.s  Arch.  Bd. 
Lxx.  (1877),  S.  72. 

*  Jaffe',  loc.  cit.  Ortweiler,  Mittheil.  d.  Wurzburg.  med.  K/inik.  Bd.  ii.  (1886), 
S.  153.     Gives  literature  to  date. 

5  Fr.  Muller,  Ibid.  S.  341  ;  Berl.  klin.  Wochensch.  1887,  Nr.  24.  (Results  of 
experiments  on  Cetti. ) 

6  Nencki,  Ber.  d.  d.  chem..  Gexell.  Bd.  vii.  (1874),  S.  1593.  See  also  Abst.  in 
Maly's  Jahresb.  1876,  S.  31.     Weyl,  Zt.  f.  physiol.  Chem.  Bd.  i.  (1877),  S   339. 

T  Walchli,  Jn.  f.  prakt.  Chem.  '(N.F.),  Bd.  xvir.  (1878),  S.  71. 


200  INDIGO-BLUE. 

in  urine.^  When  warmed  in  aqueous  solution  with  hydrochloric 
acid  it  decomposes  into  indoxyl  and  potassium  sulphate  :  — 

CgHeN .  0  .  SO2 .  OK  +  H2O  =  CsHeN  (OH)  +  KHSO,. 

Indoxyl  by  oxidation  is  converted  into  indigo-blue  :  — 

2C8H6N  (OH)  +  02  =  C^eHioNaOa  +  2H2O. 

The  blue  colouration  which  results  from  the  above  reaction  affords 
the  one  test  for  the  presence  of  indican  in  urine.  The  test  is 
applied  as  follows  (Jaffd).  A  small  volume  of  urine  (10  c.c.)  is 
mixed  with  an  equal  volume  of  strong  hydrochloric  acid  and 
2 — 3  c.c.  of  chloroform.  A  strong  solution  of  chloride  of  lime 
is  then  added  drop  by  drop,  shaking  after  the  addition  of  each 
drop.  If  indican  is  present  the  layer  of  chloroform  which  settles 
on  standing  will  be  coloured  more  or  less  brilliantly  blue  accord- 
ing to  the  amount  of  indican  in  the  urine.^  The  formation  of 
indigo-blue  is  also  the  basis  for  the  quantitative  estimation  of 
indican.  The  latter  is  converted  into  indigo-blue  by  oxidation 
and  the  indigo-blue  is  estimated  either  by  weighing  or  colori- 
metrically  or  spectrophotometrically.^ 

3.    Indigo-blue.     CieHjoN^O,. 

It  is  formed,  as  stated  above,  from  indican,  and  gives  rise  to  the 
bluish  colour  sometimes  observed  in  sweat  and  urine. 

It  may,  by  slow  formation  from  indican,  be  obtained  in  fine 
crystals ;  these  are  insoluble  in  water,  slightly  soluble,  with  a 
faint  violet  colour,  in  alcohol  and  in  ether.  Chloroform  dissolves 
them  to  a  slight  extent,  as  also  does  benzol.  Indigo  is  soluble  in 
strong  sulphuric  acid,  forming  at  the  same  time  two  compounds 
with  the  acid,  indigo  mono-  and  di-sulphonic  acids.  The  sodium 
salts  of  these  acids  are  soluble  in  water  and,  when  mixed  with 
sodium  sulphate,  constitute  the  crude  '  indigocarmine '  of  com- 
merce, and  in  a  purer  form  the  sulphindigotate  of  soda  used  in 
certain  experiments  on  the  nature  of  the  excretory  activity  of  the 
kidney  and  other  glands  (see  §  416).  These  soluble  sulphonates 
give  an  absorption  band  in  the  spectrum  which  lies  to  the  red  side 
of  and  close  to  the  D  line.     This  may  be  used  to  detect  indigo. 

Indigo  as  ordinarily  seen  possesses  a  pure  blue  colour  ;  it  leaves 
a  reddish  copper-coloured  mark  when  pressed  with  a  hard  body, 
and  the  crystals  exhibit  the  same  colour  if  seen  in  reflected  light. 

Treated  with  reducing  agents  in  strongly  alkaline  solution  in- 

i  For  its  isolation  and  preparation  from  urine  see  Baumann  u.  Brieger,  Zt. 
f.  physiol.  Chem.  Bd.  ill.  (1879),  S.  2.54.  See  also  Baumann  u.  Tiemanu,  Ber. 
d.  deutsch.  Chem.  Gesell.  xii.  (1879),  Sn.  1098,  1192  ;  and  xiii.  (1880),  S.  408. 

^  Jaffe',  Pfliiger's  Arch.  Bd.  iii.  (1870),  S.  448.  Cf.  Senator,  Centralb.  f.  d.  med. 
Wiss.  1877,  S.  357. 

^  For  details  and  literature  see  Neubauer  u.  Vogel,  Die  Harnanalyse,  1890, 
S.  492. 


CHEMICAL   BASIS    OF  THE   ANIMAL   BODY.        201 

digo  is  decolourised,  being  reduced  to  indigo-white.  The  latter 
contains  two  atoms  of  hydrogen  more  than  indigo,  is  reconverted 
into  indigo-blue  by  exposure  to  the  air,  and  thus  provides  a 
convenient  reaction  for  the  detection  of  either  indigo  or  of  re- 
ducing substances  such  as  dextrose. 

4.     Skatol.     C9H9K  CeH^;^        ^CH.      Methyl-indol. 

CH3 

Skatol  was  first  noticed  and  definitely  described  by  Brieger  as 
occurring  in  human  faeces  together  with  indol,  the  latter  usually 
being  less  in  amount  than  the  former.  ^  Later  researches  have 
shown  that  the  conditions  of  its  production  are  in  general  the 
same  as  those  for  the  formation  of  indol,  so  that  the  two  sub- 
stances occur  mixed  in  variable  proportions  among  the  products 
of  the  putrefactive  decomposition  of  proteids  ^  or  brain-substance  ^ 
and  of  the  action  of  caustic  potash  at  high  temperatures  on  pro- 
teids.* In  the  former  case  it  appears  to  be  produced  at  a  later 
stage  than  is  indol,  so  that  on  the  whole  it  is  most  preponderant 
the  longer  the  putrefactive  change  is  allowed  to  proceed.  Its 
presence  in  the  fseces  is  thus  due  to  causes  similar  to  those  which 
account  for  the  presence  of  indol,  and  the  resemblance  is  further 
shown  by  the  fact  that  a  portion  of  the  first-formed  skatol  is 
absorbed,  oxidised,  and  appears  externally  in  the  urine  as  skatoxyl- 
sulphuric  acid  (^see  helow}. 

Skatol  is  formed  in  small  quantities  during  the  preparation  of  indol 
by  reduction  from  indigo.^  It  may  be  partly  converted  into  indol  by 
passing  its  vapours  through  a  red-hot  porcelain  tube.®  The  consti- 
tution of  skatol  was  foreshadowed  by  its  preparation  from  the  barium 
salts  of  ortho-nitrocuminic  acid,  (CH3)2CH  .  CeHg  (NO^)  .  COOH  ^  and 
clearly  proved  by  its  synthetic  production  from  propjdidene-phenylhy- 
drazin  CgHg .  NH  .  jST  =  CH  .  CHg  .  CH3,  the  product  of  the  action  of 
propionic  aldehyde  (CHg  .  CHg .  COH)  on  phenylhydrazin  (CgHs  .  NH. 

1  Ber.  d.  d.  chem.  Gesell.  Bd.  x.  (1877),  S.  1027.  Jn.  f.  prakt.  Chem.  (N.F.), 
Bd.  XVII.  (1878),  S.  124.  A  closely  similar,  if  not  identical,  substance  had 
previously  been  noticed,  but  not  clearly  characterised,  by  Nencki,  as  among  the 
products  of  the  putrefactive  decomposition  of  gelatin,  and  by  Se'cretan  among  those 
of  a  similar  decomposition  of  proteids.     See  Maly's  Jahresh.  1876,  Sn.  31,  39. 

-2  Nencki,  Ceniralb.  f.  d.  med.  Wiss.  1878,  S.  849.  E.  u.  H.  Salkowski,  Ber. 
d.  d.  chem.  Gesell.  Bd.  xii.  (1879),  S.  648.  Zt.  f.  phijsiol.  Chem.  Bd.  viii.  (1884), 
S.  417 — 466.     Contains  very  full  references  to  previous  literature. 

3  "Nencki,  Ibid.  Bd.  iv.  (1880),  S.  371.  Stockly,  Jn.  f.  prakt.  Chem.  (N.F.), 
Bd.  XXIV.  (1881),  S.  17. 

*  Nencki,  Jn.f.  prakt.  Chem.  (N.F.),  Bd.  xvii.  (1878),  S.  97. 

5  Baeyer,  Ber.  d.  d.  chem.  Gesell.  Bd.  xiii.  (1886),  S.  2339. 

6  Fileti,  Gazz.  Chim.  T.  xiii.  (1883),  p.  378.  See  abstr.  in  Ber.  d.  d.  chem. 
Gesell.  1883,  S.  2928. 

1  Fileti,  loc.  cit.  p.  356.     Abst.  loc.  cit.,  S.  2927. 


202  SKATOXYL-SULPHURIC   ACID. 

NHg).     When  this  substance  is  heated  with  zinc  chloride  it  loses  NH3 
and  yields  skatol  ^ 

C6H4(         )CH. 

^  G  y 

CH3. 

Since  the  condition  of  the  occurrence  and  formation  of  skatol 
are  on  the  whole  the  same  as  those  for  indol,  and  since  these  sub- 
stances further  resemble  each  other  in  being  both  volatile  and 
hence  passing  over  in  the  vapours  arising  from  their  heated  solu- 
tions, the  method  previously  described  for  the  preparation  of  indol 
from  putrefactive  products  may  be  applied  for  the  preparation  of 
skatol.  The  separation  of  the  two  depends  chiefly  on  the  fact 
that  skatol  is  much  less  soluble  in  water  than  is  indol,  so  that  if 
the  mixed  substances  are  dissolved  in  a  minimal  amount  of  ab- 
solute alcohol,  then  on  the  addition  of  8  — 10  volumes  of  water, 
indol  remains  in  solution  while  skatol  is  precipitated.^  Skatol  is 
unaffected  by  being  boiled  with  moderately  strong  caustic  soda, 
whereas  indol  is  decomposed.  This  difference  in  behaviour  to 
caustic  alkalis  provides  a  further  means  by  which  the  former 
may  be  obtained  free  from  the  latter. 

Skatol  is  a  crystalline  substance  which  melts  when  heated  to 
93°,  and  has  a  powerfully  unpleasant  odour,  somewhat  like  that 
of  indol. 

Reactions.  Many  of  the  reactions  of  skatol  resemble  so  closely 
those  of  indol  that  they  provide  no  means  for  distinguishing  be- 
tween the  two  substances,  Skatol  is  however  characterised  by 
yielding  only  a  milky  opacity  instead  of  a  red  colouration  on  the 
addition  of  fuming  nitric  acid  to  its  aqueous  solutions  (see  siib  in- 
dol), in  not  giving  the  reaction  with  a  strip  of  pine-wood  dipped 
in  hydrochloric  acid  which  indol  does,^  by  its  lesser  solubility  in 
water  and  greater  resistance  to  the  action  of  caustic  soda. 

5.     Skatoxyl-sulphuric  acid,     CgHgN .  0  .  SO2OH. 

The  close  relationship  between  indol  and  skatol  is  further 
shown  by  the  fact  that  the  latter,  like  the  former,  after  absorp- 
tion from  the  alimentary  canal  is  oxidised,  the  product  being 
skatoxyl  CgHgN .  OH,  which  unites,  as  does  indoxyl,  with  the 
elements  of  sulphuric  acid  and  leaves  the  body  in  the  urine  as  a 
potassium  salt  of  the  above  acid.*     This  salt  may  be  isolated  from 

1  B.  Fischer,  Ber.  d.  d.  chem.  Gesell.  Bd.  xix.  (1886),  S.  1563.  Liebig's  Ann. 
Bd,  ccxxxvi.  (1886),  S.  116. 

2  Brieger,  Ber.  d.  d.  chem.  Gesell.  Bd.  xii.  (1879),  S.  1985.  Zt.  f.  phi/siol.  Chem, 
Bd.  IV.  (1880),  S.  414. 

3  When,  however,  a  strip  of  pine-wood  is  dipped  into  an  alcoholic  solution  of 
skatol  and  subsequently  into  strong  hydrochloric  acid,  it  is  coloured  first  crimson, 
which  turns  to  bluish  violet.     Fischer,  Liebig's  Ann.  Bd.  ccxxxvi.  (1886),  S.  140. 

*  Brieger,  Zt.  f.  physiol.  Chem.  Bd,  iv.  (1880),  S.  414.  Baumann  u.  Brieger, 
Ihid^  Bd.  III.  (1879),  S.  255.     G.  Hoppe-Seyler,  Ibid.  Bd.  vii.  (1883),  S,  423. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.        203 

urine  by  methods  similar  to  those  used  for  the  preparation  of 
indoxyl-sulphuric  acid. 

Our  knowledge  of  the  quantitative  formation  of  skatol  in  the 
alimentary  canal  and  of  its  relationship  to  the  simultaneous  pro- 
duction of  indol  is  far  less  complete  than  is  that  respecting  the 
latter  substance.  Notwithstanding  the  close  chemical  relation- 
ship of  the  two  it  appears  that  their  physiological  behaviour  is 
markedly  different.  In  the  first  place  it  seems  that  the  absorp- 
tion of  skatol  is  less  complete  than  that  of  indol,  since  it  pre- 
ponderates in  the  normal  faeces :  ^  in  accordance  with  this  but 
little  of  its  ethereal  sulphate  is  found  normally  in  urine.^  Fur- 
ther, whereas  by  the  ingestion  of  indol  nearly  the  whole  of  the 
sulphates  of  the  urine  may  be  converted  into  the  ethereal  com- 
pound with  indoxyl,  when  skatol  (synthetically  prepared)  is 
similarly  employed  a  large  part  reappears  in  the  faeces ;  and 
although  at  first  the  ethereal  sulphates  are  increased,  they  sub- 
sequently diminish  even  with  continued  injection  of  skatol,  and 
are  stated  to  finally  disappear.  Indoxyl-sulphuric  acid  may  be 
regarded  as  a  urinary  chromogen,  since  it  yields  a  pigment,  indigo, 
by  oxidational  decomposition ;  so  also  may  skatoxyl-sulphuric 
acid,  but  it  is  found  that  the  amount  of  pigment-forming  mate- 
rial specifically  present  in  the  urine  of  a  dog  fed  with  skatol  is 
not  so  directly  proportional  to  the  amount  of  skatoxyl-sulphuric 
acid  as  it  is  to  the  similar  compound  of  indoxyl  when  indol  is 
administered.  It  has  been  suggested  that  a  large  part  of  the 
skatolic  chromogen  exists  as  a  compound  of  skatoxyl  and  glycu- 
ronic  acid.^  When  Jaffa's  test  (see  p.  200)  for  urinary  indican  is 
applied  to  urine  which  contains  skatoxyl  compounds  the  results 
obtained  are  as  follows.  The  urine  turns  dark  red  or  violet  on 
the  addition  of  hydrochloric  acid,  bright  crimson  on  the  addition 
of  nitric  acid,  and  a  similar  colour  is  obtained  if  it  is  warmed 
with  hydrochloric  acid  and  ferric  chloride.  The  colouring  mat- 
ter thus  obtained  is  probably  formed  from  the  skatoxyl  (not 
known  in  the  free  state),  and  by  reduction  may  be  made  to 
yield  skatoL 

Skatol  has  recent!}^  been  described  as  occurring  in  a  vegetable  tis- 
sue, namely  the  wood  of  an  East  Indian  tree,  Celtis  reticulosa.^ 

1  It  is  absent  from  the  fasces  of  the  dog. 

2  The  chief  record  of  its  occurrence  is  in  a  case  of  diabetes  mellitus  with 
gastric  disturbance.     Otto,  Pfluger's  J.rc^.  Bd.  xxxiii   (1884),  S.  607. 

'■^  Mester,  Zt.  f.  phijsioL  Chem.  Bd.  xii.  (1888),  S.  130.  A  similar  compound  of 
indoxyl  with  glycuronic  acid  has  been  described.  Schmiedeberg,  Arch.  f.  exp.  Path, 
u.  Pharm.  Bd.  xiv.  (1881),  S.  306.  To  complete  the  literature  of  this  substance 
see  E.  Salkowski,  Zt.  f.  physiol.  Chem.  Bd.  viii.  S.  417  ;  ix.  (1884),  Sn.  8,  23. 

4  Dunstan,  Pharm.  JL  Vol.  xix.  (1889),  p.  1010.  Ber.  cl.  d.  chem.  Gesell. 
(Referate),  Bd.  xxii.  (1889),  S.  441.     Proc.  Roy.  Soc.  Vol,  xlvi.  (1889),  p.  211. 


204  PTOMAINES. 


The  Ptomaines. 


The  now  extensive  literature  of  these  substances  may  be  most  con- 
veniently and  inclusively  indicated  by  reference  to  the  following 
publications.  Selmi  (to  whom  the  name  ptomaine  is  due),  Sulle 
ptomaine  od  alcaloidi  cadaverici.  Bologna,  1878.  Gautier,  Compt. 
Bend.  T.  xciv.  (1882),  p.  1119.  Guareschi  e  Mosso,  Arch.  ital.  de 
Biol.  T.  II.  (1883),  p.  367;  in.  (1883),  p.  241.  Abstr.  in  Jn.  f. 
prakt.  Chem.  (N.F.),  Bd.  xxvii.  S.  425;  xxviii.  S.  504.  Brieger, 
Zt.  f.  physiol.  Chem.  Bd.  vii.  (1883),  S.  274.  Ber.  d.  d.  chem.  Gesell. 
Bd.  XVI.  (1883),  Sn.  1186,  1405.  E.  u.  H.  Salkowski,  Ibid.  S.  1191. 
Brieger,  Ibid.  Bd.  xvii.  (1884),  Sn.  515,  1137,  2741;  xix.  (1886),  S. 
3119.  Ueber  Ptomaine,  i.,  n.  Berlin,  1885;  in.  1886:  gives  litera- 
ture to  date.  See  resume  with  references  by  0.  Schultz,  Biol.  Centralb. 
Bd.  VI.  (1886-87),  Sn.  685,  726,  739.  Gautier,  Bull,  de  Vacad.  de 
med.  Jan.  12,  19,  1886  (largely  on  the  leukomaines).  Udranszky  u. 
Baumann,  Zt.  f.  phijsiol.  Chem.  Bd.  xiii.  (1889),  S.  562.  Brieger, 
Virchow's  Arch.  Bd.  cxv.  (1889),  S.  483.  The  last  contains  a  most 
useful  list  of  known  ptomaines,  with  empirical  and  constitutional 
formula,  name  of  discoverer  with  date  of  discovery,  sources,  action, 
and  characteristic  reactions. 

Although  the  substance  to  which  the  above  name  has  been 
given  (from  7rTw/x,a,  a  corpse)  are  now  known  to  belong  chiefly  to 
the  class  of  compounds  called  amines,^  so  that  they  provide  no 
chemical  sequence  to  the  bodies  previously  described,  their  charac- 
teristic production  during  the  putrefactive  decomposition  of  animal 
tissues  seems  to  make  this  a  suitable  place  for  treating  of  them. 

The  ptomaines  as  a  group  may  be  said  to  closely  resemble  the 
class  of  substances  long  known  under  the  name  of  alkaloids  and 
obtained  from  plant  tissues.  The  resemblance  is  shown  not 
merely  in  their  chemical  constitution,  but  more  obviously  in  their 
similar  solubilities  in  various  fluids,  in  their  general  behaviour 
towards  reagents,  and  in  some  cases  even  in  their  specific  reactions, 
and  more  especially  in  their  frequently  powerful  (poisonous)  action 
on  the  animal  organism,  the  actions  of  certain  ptomaines  being 
almost  identical  with  those  of  certain  vegetable  alkaloids.  The 
ptomaines  may  therefore  be  regarded  as  alkaloids  of  animal  origin. 
The  close  similarity  of  the  two  classes  of  substances  has  hence 
endowed  the  ptomaines  with  very  considerable  interest  from  a 
medico-legal  point  of  view,  in  virtue  of  the  not  infrequent  use  of 
the  vegetable-alkaloids  for  criminal  purposes  and  the  now  obvious 
possibility  that  the  detection  of  alkaloids  in  the  corpse  may  afford 
no  reliable  information  as  to  the  administration  of  the  same  dur- 

1  An  amine  is,  strictly  speaking,  a  compound  ammonia  in  which  one  or  more 
atoms  of  hydrogen  have  been  replaced  by  some  oxygen-free  radical.  Several  of  the 
ptomaines,  however,  contain  oxygen  in  their  molecule,  as  do  also  many  of  the  vegetable 
alkaloids.  The  constitution  of  those  ptomaines  which  contain  oxygen  has  not  in 
most  cases  as  yet  been  as  definitely  determined  as  has  that  of  those  which  contain 
none. 


CHEMICAL  BASIS   OF   THE   ANIMAL   BODY.        205 

ing  life.^  They  are  further  of  considerable  and  increasing  patho- 
logical interest,  and  that  from  two  points  of  view.  In  the  first 
place,  as  products  of  the  general  putrefactive  changes  which 
animal  tissues  undergo,  they  may  account  for  the  severe  symptoms 
and  not  infrequent  death  which  results  from  the  consumption  as 
food  of  fish,  sausages,  and  tinned-meats.  In  the  second  there 
appears  to  be  increasing  evidence  of  the  formation  of  special 
ptomaines  by  the  organisms  characteristic  of  specific  diseases,  so 
that  the  pathological  conditions  may  be  due  rather  to  the  products 
formed  by  the  organisms  than  to  the  organisms  themselves  directly, 
a  possibility  of  no  small  importance  in  the  light  of  recent  prophy- 
lactic research. 

While  the  general  reactions  of  the  ptomaines  place  them,  as 
already  stated,  side  by  side  with  the  vegetable  alkaloids,  their 
specific  reactions  and  properties  exhibit  considerable  differences 
both  in  comparison  with  each  other  and  with  those  of  the  alka- 
loids^ Some  are  liquid  and  highly  volatile  so  that  they  pass  off 
readily  during  distillation  of  their  aqueous  solutions,  others  are 
liquid  and  non-volatile,  others  again  solid  and  crystalline.  They 
exhibit  equally  marked  differences  in  their  solubilities.  Thus 
neither  benzol  nor  petroleum-ether  will  extract  them  from  their 
acid  aqueous  solution.  Ether  on  the  other  hand  dissolves  out  a 
few  of  the  ptomaines  from  an  acid  solution  and  a  large  majority 
from  an  alkaline  solution.  Some  are  more  particularly  soluble  in 
chloroform,  a  few  are  insoluble  in  any  of  these  reagents.  Amyl- 
alcohol  is  the  one  reagent  in  which  as  a  class  they  appear  to  be 
almost  generally  soluble  (Brieger).  Their  behaviour  with  the 
usual  alkaloidal  precipitants  (mercuric  and  platinic  chlorides, 
tannic  acid,  the  double  iodides  of  potassium  with  mercury  and 
other  metals,  &c.)  is  equally  varied.  They  are  all  precipitated  by 
phospho-molybdic  acid,  and  most  of  them  yield  crystalline  com- 
pounds with  a  solution  of  iodine  in  hydriodic  acid.  Possessed  of 
an  alkaline  reaction  they  further  act  as  powerful  reducing  agents, 
many  of  them  converting  ferri-  into  ferrocyanides,  the  reduction 
being  evidenced  by  the  formation  of  Prussian  blue  on  the  simulta- 
neous addition  of  ferric  chloride.  This  property  is  however 
possessed  by  many  vegetable  alkaloids  and  not  by  every  ptomaine  ; 
it  cannot  therefore  be  regarded  as  a  specific  class-reaction  for  these 
substances  (Brieger,  Gautier).  Some  of  the  ptomaines  (Toxines) 
are  extraordinarily  poisonous,  producing  effects  which  are  fre- 
quently specific,  but  in  many  cases  similar  to  those  of  certain 
vegetable  alkaloids.     Others  again  are  quite  inert. 

The  separation  of  the  ptomaines,  as  of  the  vegetable  alkaloids, 
involves  the  application  of  methods  (Stas-Otto's,  Brieger's)^  which 

1  For  cases  in  point  see  Husemann,  Arch.  d.  Pharm.  (Keihe  3)  Bde.  xvi.  xvii. 
XIX.  XX.  (1882),  XXI.  (1883),  Sn.  169,  327,  187,  270,  401,  u.  481. 

2  Otto,  Anieit.  zur  Ausmittelunq  d.  Gifte,  Aufl.  6,  1884,  S.  88  et  seq. 

3  Unters.  iib.  Ptomaine,  ii.  1885,  S.  52. 


206  PTOMAINES. 

admit  of  no  suitably  brief  description.  The  principle  involved  in 
each  consists  in  preparing  a  concentrated  alcoholic,  ethereal,  or 
chloroformic  extract  of  the  mother-substance,  and  from  this 
crystalline  compounds  of  the  alkaloids  are  prepared  by  the  addi- 
tion of  suitable  reagents.^  A  further  means  for  their  final  separa- 
tion consists  in  the  formation  of  benzoylated  compounds  which  are 
insoluble  in  water.^ 

Alkaloidal  substances,  some  poisonous,  others  inert,  may  also  be 
obtained  both  from  normal  but  more  particularly  from  pathological 


urmes 


The  first  distinct  evidence  that  the  poisonous  properties  of  cer- 
tain (septic)  fluids  might  be  due  to  a  specific  chemical  substance 
rather  than  necessarily  to  the  action  of  organisms  in  those  fluids 
is  apparently  due  to  Panum,  who  seems  to  have  dealt  with  a 
septic  alkaloid  in  a  very  pure  form,  although  he  did  not  definitely 
characterise  it  as  a  chemical  substance.*  This  was  followed  by  a 
series  of  observations  all  tending  in  the  same  direction,  but  none 
of  the  observers  obtained  the  supposed  specifically  toxic  substances 
in  forms  which  enabled  them  to  be  spoken  of  as  chemical  in- 
dividuals until  Nencki  in  1876  ^  isolated  from  the  products  of 
the  pancreatic  putrefaction  of  gelatin  a  well-crystallised  base 
CgHiiN,    to    which    he     assigned     the     constitutional    formula 

/CH3 
CgHj  —  CH  and    hence    the    name    isophenyl-ethvlamin. 

Since  then  the  ptomaines  have  been  in  most  cases  fairly  definitely 
and  in  some  cases  absolutely  characterised  as  regards  their  chemi- 
cal composition  and  constitution.  They  belong  typically  to  the 
class  of  substances  known  as  amines  and  are  in  many  cases  dia- 
mines. Two  of  the  most  clearly  defined  ptomaines  are  cadaverin 
and  putrescin.  These  are  found  in  corpses  which  have  undergone 
putrefactive  decomposition,  cadaverin  appearing  in  the  earlier 
stages  of  putrefaction,  and  putrescin  preponderating  in  the  later. 
The  latter  is  largely  present  in  putrid  herrings.^  The  former  is 
identical  with  pentamethylen-diamine  ]SrH2  (CH2)5NH2."  The 
latter  has  been  shown  to  have  the  constitution  of  tetramethylen- 
diamine  IsTHg  (CH2)4NH2.  They  have  both  recently  been  obtained 
as  conspicuous  constituents  of  urine  from  a  case  of  cystinuria,  and 

1  For  description  of  these  methods  see  Halliburton,  Chem.  Physiol,  and  Pathol. 
1891,  p.  175.     Otto,  loc.  cit.  S.  103. 

2  Udranszky  u.  Baumann,  Zt-  f.  physiol.  Chem.  Bd.  xiii.  (1889),  S.  562. 

3  For  details  and  literature  see  Neubauer  u.  Vogel,  Anal.  d.  Hams.  1890,  S  241 
et  seq. 

*  Published  originally  in  Danish  in  Bibliotheh  f.  Ldqer,  April,  1856,  S,  253, 
Fully  abstracted  in  Schmidt's  Jahrbucher  d.  ges.  Med.  Bd.  ci.  (1859),  S.  213,  and 
republished  in  Virchow's  Arch.  Bd.  lx.  (1874),  S.  301,  with  literature  up  to  date. 

^  Ueb,  d.  Zersetz.  d.  Gelatine  u.  s.  w.  Bern,  1876.  See  \2itev  Jn.  f.  prakt.  Chem. 
Bd.  XXVI.  (1882),  S.  47. 

6  Bocklisch,  Ber.  d.  d.  chem.  GeselL  Bd.  xviii.  (1885),  Sn.  86,  1922;  xx.  (1887), 
S.  1441. 

^  Ladenburg,  Ibid.  Bd.  xix.  (1886),  S.  2585. 


CHEMICAL   BASIS    OF  THE  ANIMAL   BODY.        207 

appear  to  owe  their  origin  to  putrefactive  processes  occurring  in 
the  intestine. 1  They  are  both  somewhat  viscid  fluids  which 
crystallise  at  low  temperatures,  and  yield  readily  crystallisable 
compounds  with  acids  and  salts  of  gold,  platinum,  and  mercury. 
Their  benzoyl  compounds  are  insoluble  m  water  and  hence  afford 
a  convenient  means  for  their  separation.  Cholin  and  the  highly 
toxic  neurin,  which  really  belong  to  this  class,  have  already  been 
described.     (See  above  pp.  135,  136.) 

Leukomaines.  This  name  has  been  applied  by  Gautier^  to  those 
basic  (alkaloidal)  substances  which  occur  in  living  tissues  and  are 
to  be  regarded  as  products  of  their  normal  metabolism  and  thus 
distinct  from  ptomaines.  They  are  obtained  by  extracting  finely 
minced  ox-flesh  with  an  extremely  dilute  aqueous  solution  of 
oxalic  acid.  According  to  Gautier  this  extract  may  contain  the 
following  six  bases :  Xanthokreatinin,  C5H10N4O ;  Chrysokreatinin, 
C5H8N4O  ,  Amphikreatinin,  C8H19N7O4 ,  Pseudoxanthin,  C4H5]Sr50 
and  two,  as  yet  unnamed,  with  the  composition  C11H24N10O5  and 
CiaHasNuOs  respectively.  They  probably  stand  in  close  relation- 
ship to  paraxanthin,  C7H8N4O0,  heteroxanthin,  C6H6N4O2,  and 
adenin  C5H5N5  (see  above,  p.  181),  and  it  is  interesting  to  note 
that  comparing  the  formulae  of  the  leukomaines  with  each  other 
and  with  those  of  kreatinin  C4H7N3O  and  kreatin  C4H9N3O2  they 
are  found  to  differ  in  several  cases  by  the  group  CNH. 

The  leukomaines  are  regarded  by  Gautier  as  feebly  toxic  alka- 
loidal products  of  metabolism  from  which  the  organism  is 
normally  freed  either  by  their  excretion,  since  they  are  found  in 
urine  (see  above),  or  by  destructive  oxidation,  and  it  has  further 
been  suggested  that  their  abnormal  retention  in  the  economy  may 
be  the  cause  of  certain  obscure  pathological  conditions.^ 


The  Bii^e-Acids. 

1.     Cholalic  (or  cholic)  acid.     C24H40O5. 

To  avoid  confusion  the  term  '  cholic '  should  be  in  all  cases  used  as 
synonymous  with  '  cholalic.  '  Demarcay,  who  first  described  cholalic 
acid  as  a  product  of  the  decomposition  of  bile-acids,  gave  it  the  name 
of  cholic  acid.'*  The  name  'cholalic'  is  perhaps  the  better,  since  it 
indicates  the  method  by  which  the  bile-acids  are  decomposed  during 
its  preparation,  viz.  by  treatment   with  alkalis.      The  name  '  cholic  ' 

1  Udranszky  u.  Baumann,  he.  cit.  See  also  Stadthagen  u  Brieger,  Virchow's 
Arch.  Bd.  oxv.  (1889),  S.  490. 

^  Siir  les  alcaloides  derives  de  la  destruction  bncte'rlenne  oti  phi/siologique  des 
tissus  anhnaux.  Paris,  1886.  Bull,  de  I'acad.  de  me'd.  Jan.  12,  19,  1886.  The  name 
is  derived  from  KevKuixa,  occasionally  used  to  denote  white  of  egg,  and  hence  to 
indicate  their  origin  from  proteids. 

3  Cf.  Bouchard,  Compt.  Rend.  T.  cii.  (1886),  pp.  669,  727.  1127, 

*  Liebig's  Ann.  Bd.  xxvii.  (1838),  S.  270. 


208  CHOLALIC  ACID. 

was  first  applied  by  Gmelin  ^  and  siibsequently  by  Strecker  ^  to  the 
acid  which  is  now  always  known  as  glycocholic.  The  acid  now  known 
as  taurocholic  was  originally  called  'choleic'  by  Demarcay,  and  the 
same  name  has  been  quite  recently  used  to  denote  an  acid  (C25H42O4) 
closely  related  to  cholalic  acid  (see  below). 

This  acid  occurs  in  traces  as  a  product  of  the  decomposition  of 
the  bile-acids  in  the  small  intestine,  in  larger  quantities  in  the 
contents  of  the  large  intestine,  and  in  the  faeces  of  man  and  many 
animals.  In  icterus  the  urine  is  also  stated  to  frequently  contain 
traces  of  this  acid.  Its  principal  interest  lies  in  its  being  the 
starting-point,  by  its  union  with  glycin  or  taurin,  for  the  acids 
which,  as  sodium  salts,  exist  characteristically  in  bile  (see 
below). 

Owing  to  the  readiness  with  which  ox-bile  can  be  obtained  in 
large  quantities,  this  has  been  most  frequently  used  for  the  prep- 
aration of  cholalic  acid,  whose  properties  as  usually  given  hence 
refer  to  the  acid  as  obtained  from  this  source.  More  recent  re- 
searches have  however  demonstrated  comparatively  unimportant 
but  still  distinct  differences  in  the  composition  and  properties  of 
the  acid  as  it  occurs  in  the  bile-acids  of  different  animals.  The 
description  of  the  acid  which  here  follows  refers  to  that  form 
which  is  obtained  from  ox-bile. 

Preparation.  This  depends  upon  the  decomposition  of  the 
bile-acids  (glycocholic  and  taurocholic)  by  means  of  alkalis  at 
boiling  temperature.  It  is  not  however  necessary  to  employ  the 
purified  acids  for  this  purpose  since  the  raw  bile  suffices.  The  bile 
is  boiled  for  twenty-four  hours  with  as  much  caustic  baryta  as  it 
will  hold  in  solution,  concentration  during  this  operation  being 
avoided  by  means  of  a  condenser  attached  to  the  mouth  of  the 
flask.  When  the  decomposition  is  complete  the  fluid  is  filtered 
while  still  hot,  and  the  filtrate  concentrated  until  crystals,  con- 
sisting of  the  barium  salt  of  the  acid,  are  copiously  formed.  These 
are  then  purified  by  recrystallisation  from  boiling  water  and  de- 
composed by  the  addition  of  hydrochloric  acid.  The  free  cholalic 
acid  is  finally  obtained  in  a  pure  form  by  solution  in  a  small 
volume  of  boiling  alcohol  from  which  it  separates  out  in  the 
crystalline  form  on  cooling. 

As  thus  prepared  the  acid  possesses  the  following  properties. 
The  crystals  obtained  from  hot  alcoholic  solutions  are  usually  in 
the  form  of  large  rhombic  tetrahedra  or  octahedra,  containing  2^ 
molecules  of  water  of  crystallisation  which  may  be  driven  off  by 
heating  to  100°  C.  The  crystals  are  but  slightly  soluble  (1  in 
750)  either  in  water,  even  when  boiling,  or  in  ether,  but  readily 
soluble  in  alcohol.  This  acid  may  also  be  obtained  in  an  amor- 
phous form  by  concentrating  its  solutions  to  dryness,  and  is  now 

^  Die  Verdaunng  nach  Versuchen,  1826. 
2  Liebig's  Ann.  Bd.  lxv.  (1848),  S.  1. 


CHEMICAL  BASIS   OF  THE   AKIMAL  BODY.        209 

less  insoluble  than  when  crystallised.  If  the  amorphous  acid  is 
dissolved  in  ether  it  may  be  separated  out  by  evaporation  in  four 
or  six-sided  prisms  which  are  anhydrous.  When  the  sodium  salt 
of  cholalic  acid  is  decomposed  under  ether  by  the  addition  of 
hydrochloric  acid,  the  acid  may  be  obtained  in  rhombic  plates 
containing  one  molecule  of  water.  The  alkali  and  barium  salts  of 
cholalic  acid  are  soluble  in  water  and  in  alcohol,  especially  when 
warm,  and  yield,  like  the  free  acid,  dextro-rotatory  solutions. 
For  solutions  of  the  anhydrous  acid  (a)jy  =  -\- 50°.  When  crys- 
tallised with  2^  H2O,  {a)j)  =  -f-  35°.  In  alcoholic  solutions  of  the 
sodium  salt  (a)u  =  +31°'4  (Hoppe-Seyler). 

The  constitution  of  cholalic  acid  is  scarcely  as  yet  definitely 

rcooH 

known,  but  may  be  represented  by  CgoHgj  -|  (CH20H)2.i      It  yields 

(  CHOH 
with  iodine  a  compound  which,  like  that  resulting  from  the  inter- 
action of  iodine  and  starch,  possesses  a  brilliantly  blue  colour  and 
is  specifically  distinctive,  since  it  cannot  be  obtained  either  from 
the  bile-acids  or  choleic  acid  (see  below)  or  the  products  of  the 
decomposition  of  cholalic  acid.^ 

When  cholalic  acid  is  prepared  from  human  bile  it  exhibits 
certain  differences,  more  especially  as  regards  the  lesser  solubili- 
ties of  its  alkali  and  barium  salts,  which  led  to  its  being  regarded 
as  distinct  from  that  obtained  from  ox-bile,  and  hence  it  was  called 
anthropocholalic  acid.  It  appears  however  that  the  bulk  of  the 
acid  is  identical  with  that  from  ox-bile,  the  slight  difference  being 
due  to  an  admixture  with  another  acid  either  choleic,  as  was  first 
supposed,  or  fellic.^ 

Choleic  acid,  C25H42O4.  Is  obtained  in  small  amounts  mixed  with 
cholalic  acid  dviring  the  preparation  of  the  latter  from  ox-bile.  It 
differs  from  cholalic  acid  in  the  solubilitj"  of  its  salts  and  the  products 
of  its  oxidational  decomposition.'' 

Fellic  acid,^  C23H40O4.  Obtained  in  small  amounts  from  human 
bile  during  the  preparation  of  ordinary  cholalic  acid.  It  is  character- 
ised by  the  extreme  insolubility  of  its  barium  and  magnesium  salts. 
It  also  yields  a  less  brilliant  Pettenkofer  reaction  (see  below)  than 
does  cholalic  acid. 

The  bile-acids  of  the  pig  and  goose  when  decomposed  yield  forms  of 
cholalic  acid  called  respectively  hyo-cholalic  acid  C25H40O4,  and  cheno- 
cholalic  C27H44O4. 

1  Mylius,  Ber.  d.  d.  chem.  Gesell.  Bd.  xx.  (1887),  S.  1968. 

2  Mylius,  Ibid.  S.  683  and  Zt.  f.  physiol.  Chem.  Bd.  xi.  (1887),  S.  306.  See  also 
Bd.  XII.  (1888;,  S.  262. 

3  Schotten,  Zt.  f.  physiol.  Chem.  Bd.  x.  (1886),  S.  175  ;  xi.  S.  268. 
*  Latschinoff,  Ber.  d.  d.  chem.  Gesell.  Bd.  xviii.  (1885),  S.  3039. 

^  Schotten,  loc.  cit. 

14 


210  DYSLYSIN.     GLYCOCHOLIC   ACID. 

2.     Dyslysin.    C^Jl^^O^. 

When  cholalic  acid  is  heated  to  200°  C.  or  boiled  for  some  time 
in  solution  with  hydrochloric  or  sulphuric  acid  it  loses  two  mole- 
cules of  water  and  yields  a  resinous  anhydride  called  dyslysin, 
from  its  insolubility  in  water,  alcohol,  and  alkalis.  As  resulting 
from  the  dehydration  of  cholalic  acid  it  is  found  sometimes  in 
small  amount  in  the  faeces.  It  is  a  non-crystalline  substance 
which  is  soluble  in  an  excess  of  ether,  also  in  solutions  of  cholalic 
acid  or  of  its  salts.  By  treatment  with  boiling  alkalis  it  may  be 
reconverted  by  hydration  into  cholalic  acid. 

The  various  forms  of  cholalic  acid  which  may  be  prepared  from 
the  bile  of  different  animals  each  yield  a  corresponding  form  of 
dyslysin. 


3.     Glycocholic  acid.    CggH^i^O, 


This  substance  was  first  described  by  Gmelin  (1826),  by  whom 
it  was  then  named  '  cholic  acid.'  It  is  found  not  in  the  free  state 
but  as  a  sodium  salt,  chiefly  in  ox-bile  but  also  in  that  of  man, 
mixed  in  both  cases  with  a  much  smaller  and  variable  amount  of 
taurocholic  acid,  also  present  as  a  sodium  salt.  In  carnivora  it 
occurs,  if  at  all,  in  such  minute  traces  only,  that  it  may  be  said 
to  be  absent  from  the  bile  of  these  animals  ;  hence  their  bile-acid 
consists  entirely  of  taurocholic  acid.^  In  icterus  the  urine  may 
contain  small  quantities  of  glycocholic  acid. 

Preparation.  This  may  be  affected  in  several  ways,  using  ox- 
bile  as  the  source ;  of  these  the  following  is  as  convenient  as  any 
(Drechsel).^  The  bile  is  mixed  with  washed  sand  and  evaporated 
on  a  water-bath  until  it  presents  a  pulverisable  mass.  This  is 
then  extracted  in  a  flask  with  strong  boiling  alcohol  and  yields  a 
green  solution,  which  is  filtered,  decolourised  with  animal  char- 
coal, and  concentrated  to  a  sirup.  The  latter  is  then  dissolved 
in  a  minimal  quantity  of  absolute  alcohol  and  precipitated  by  an 
excess  of  ether.  The  precipitate  which  consists  of  glycocholate 
of  soda  together  with  the  corresponding  salt  of  any  taurocholic 
acid  which  is  present  in  the  bile,  is  collected,  dissolved  in  a  little 
water,  and  acidulated  with  sulphuric  acid  in  presence  of  some 
ether  as  long  as  any  precipitate  is  formed.  By  this  means  the 
acids  are  separated  from  their  sodium  salts,  and  on  standing  a 
crystalline  mass  of  glycocholic  acid  is  obtained,  practically  free 
from  taurocholic  acid,  which,  since  it  is,  unlike  the  glycocholic, 
extremely  soluble  in  cold  water,  remains  in  solution  in  the  mother 
liquor.     The  crystals  may  be  purified  by  recrystallisation  from 

1  For  earlier  references  to  the  bile-acids  of  various  animals  see  Bayer,  Zt.  f. 
pkysiol.  Chetn.  Bd.  in.  (1879),  S.  293. 

2  Anleit.  z.  Darstell.  physiol.-chem.  Prdparate,  1889,  S.  33. 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.        211 

liot  water  in  which  they  are  soluble,  separating  out  again  as  their 
solution  cools.i 

The  acid  crystallises  in  fine  glistening  needles,  which  require 
about  300  parts  of  cold  but  only  120  of  hot  water  for  their  solu- 
tion. They  are  also  very  soluble  in  alcohol,  but  practically  in- 
soluble in  ether.  The  salts  of  this  acid,  more  especially  those 
with  the  alkalis,  are  extremely  soluble  even  in  cold  water,  also 
in  alcohol,  but  very  slightly  so  if  at  all  in  ether.  Both  the  free 
acid  and  its  salts  are  dextro-rotatory :  for  the  former,  in  alcoholic 
solutions,  (a)D  =  +  29-0°,  for  the  latter  (a)^^  =+  25-7°  (Hoppe- 
Seyler).  Glycocholic  acid  is  a  compound  of  cholalic  acid  and 
glycin  (glycocoll)  or  amido-acetic  acid.  When  boiled  with  hy- 
drolysing  agents  such  as  dilute  acids  or  alkalis  it  takes  up  one 
molecule  of  water  and  is  resolved  into  its  components  :  — 

Glycin.  Cholalic  acid. 

C^eH^NOe  -f  H,0  =  CH^  (NH,)  COOH  +  C,,H,oO,. 

It  is  thus  analogous  in  constitution  to  hippuric  acid,  in  which 
glycin  is  similarly  united  to  benzoic  acid. 

If  dissolved  in  concentrated  sulphuric  acid  and  then  warmed,  glyco- 
cholic  acid  by  the  removal  of  one  molecule  of  water  yields  glj^cocholonic 
acid,  C26H4ijSr05.  The  barium  salt  of  this  last  acid  is  insoluble  in 
water,  which  fact  is  of  importance,  since  cholonic  acid  possesses  nearly 
the  same  specific  rotatory  power  as  glycocholic  acid. 

4.     Taurocholic  acid.     C^^H^NSO,. 

This  acid  is  found  to  some  extent  in  ox-bile,  and  is  more  plen- 
tifully present  in  that  of  man.  The  bile  of  the  dog  contains 
taurocholic  acid  alone,  unmixed  with  glycocholic. 

Preparation.  The  method  described  above  suffices  to  obtain 
glycocholic  acid  free  from  taurocholic.  On  the  other  hand  it  is 
not  by  any  means  easy  to  obtain  the  latter  free  from  the  former, 
for  taurocholic  acid  is  extremely  soluble  in  water  (its  crystals  are 
deliquescent)  and  this  solution  can  readily  dissolve  the  much  less 
readily  soluble  glycocholic  acid.  Hence  the  mother  liquor  from 
the  glycocholic  acid  crystals  contains  not  merely  the  taurocholic 
acid  but  some  of  the  former  acid  also.  This  difficulty  is  avoided 
by  employing  as  a  source  for  its  preparation  dog-bile  in  which 
there  is  no  glycocholic  acid.  The  bile  is  treated  as  already  de- 
scribed down  to  the  stage  at  which  the  taurocholate  of  soda  is 
precipitated  from  its  alcoholic  solution  by  an  excess  of  ether. 
The  precipitate  is  now  dissolved  in  water  and  the  acid  precipitated 
as  a  lead  salt  by  the  addition  of  ammonia  and  basic  lead-acetate. 

1  For  details  of  other  methods  some  special  work  should  be  consulted,  such  as 
Hoppe-Seyler's  Handbuch.  See  also  Malv  in  Hermann's  Handhuch  d.  PInjsiol. 
Bd.  V.  Th.  2,  S.  130.     Cf.  Mylius,  Zt.  f.  physiol.  Chem.  Bd.  xi.  (1887),  S.  231. 


212  TAUEOCHOLIC   ACID. 

This  is  next  washed,  suspended  in  alcohol,  and  decomposed  by 
sulphuretted  hydrogen.  After  removal  of  the  sulphide  of  lead 
by  filtration  the  alcoholic  filtrate  is  concentrated  and  the  tauro- 
cholic  acid  precipitated  by  an  excess  of  ether.  This  yields  a  sirupy 
mass  which  may  become  partly  crystalline  on  standing :  the 
crystals  at  once  deliquesce  on  exposure  to  the  air.i  As  dog-bile 
is  not  readily  obtainable  in  large  quantity  at  any  one  time,  it  may 
be  desirable  sometimes  to  obtain  taurocholic  acid  from  the  mother 
liquor  left  in  the  preparation  of  glycocholic  acid.  The  separation 
is  effected  by  the  addition  of  a  little  ammonia  and  normal  lead 
acetate.  This  precipitates  both  glycocholic  and  cholalic  acid,  but 
not  taurocholic.  After  the  removal  of  this  precipitate  the  tauro- 
cholic acid  is  prepared  as  already  described  by  the  addition  of 
basic  lead  acetate  to  the  filtrate. 

This  acid,  as  already  stated,  is  extremely  soluble  in  water  and 
in  alcohol,  but  not  in  ether ;  so  also  are  its  salts  with  the  excep- 
tion of  the  one  formed  on  the  addition  of  basic  lead  acetate  in 
presence  of  ammonia,  which  is  insoluble  in  water  and  in  alcohol. 
The  acid  and  its  salts  are  dextro-rotatory  ;  for  the  sodium  salt 
in  alcoholic  solution  (a)D  =  H-  24-5°.  If  dissolved  in  water  the 
rotatory  power  is  less,  and  in  this  respect  it  resembles  glycocholic 
acid. 

When  hydrolised  it  readily  takes  up  a  molecule  of  water  and  is 
decomposed  into  taurin  and  cholalic  acid  :  — 

Taurin  Cholalic  acid. 

C26H45NSO7  +  H2O  =  NH2 .  CHo .  CHo .  SOoOH  4-  C04H40O5. 

This  decomposition  may,  as  in  the  case  of  glycocholic  acid,  be 
brought  about  by  the  action  of  dilute  acids  or  alkalis,  but  even 
mere  boiling  of  an  aqueous  solution  of  the  acid  also  suffices, 
a  fact  which  demonstrates  how  unstable  a  substance  it  is, 
both  absolutely  and  as  compared  with  glycocholic  acid.  Tau- 
rocholic acid  has  not  as  yet  been  observed  in  the  urine  in 
icterus,  but  since  cholalic  acid  does  occur  together  with  glyco- 
cholic acid,  it  is  probable  that  the  non-appearance  of  taurocholic 
acid  is  due  to  its  decomposition  before  excretion  as  a  result  of  its 
instability. 

Taurocholic  acid  possesses  a  remarkable  power  of  effecting  the 
complete  precipitation  of  ordinary  proteids  from  their  solutions, 
whereas  peptones  if  present  at  the  same  time  remain  unprecipi- 
tated.  This  is  possibly  of  some  not  inconsiderable  importance  in 
connection  with  the  changes  which  proteids  undergo  in  the  small 
intestine,  since  it  leads  to  the  retention  of  the  peptones  in  a  state 
of  solution  and  hence  facilitates  their  absorption,  while  the  less 
completely  altered  proteids  are  precipitated  and  thus  further  ex- 

i  Parke,  Hoppe-Seyler's  Med.-chem.  Unters.  Hft.  1.  (1866),  S.  160. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        213 

posed  to  the  action  of  the  digestive  enzymes.^    It  is  also  possessed 
of  powerful  antiseptic  properties.^ 

The  acids  obtained  from  the  bile  of  different  animals  differ  slightly 
in  properties  and  composition,  dependently,  as  already  stated,  upon  the 
differences  between  the  several  forms  of  cholalic  acid  with  which  either 
the  glycin  or  taurin  is  respectively  united. 

Pettenkofer' s  reaction  for  Mle  acids.  ^ 

The  following  is  the  more  usual  method  of  obtaining  the  reac- 
tion. Bile,  which  may  be  very  considerably  diluted,  or  a  dilute  solu- 
tion of  bile-salts  or  acids  is  mixed  in  a  porcelain  dish  with  a  few 
drops  of  a  10  p.  c.  solution  of  cane-sugar.  Concentrated  sulphuric 
acid  is  now  added  to  the  mixture  with  constant  stirring  to  an  ex- 
tent not  exceeding  f  of  its  volume,  the  addition  of  the  acid  being 
so  regulated  that  the  temperature  of  the  mixture  is  not  allowed 
to  rise  above  70°  C.  Hereupon  a  brilliant  cherry-red  colour  makes 
its  appearance  and  rapidly  assumes  a  magnificent  purple  tint.  On 
standing  for  some  time  the  colour  becomes  darker  and  of  a  more 
distinctly  blue  tint.  The  reaction  may  also  be  obtained  by  the 
addition  of  first  the  acid  and  then  the  sugar  solution.  The  suc- 
cess of  the  test  depends  on  the  careful  avoidance  of  any  excessive 
rise  of  temperature  during  the  addition  of  the  sulphuric  acid  and 
more  especially  of  any  excess  of  sugar  which  by  being  charred  by 
the  acid  gives  a  brown  colouration  and  masks  the  typical  purple.* 
The  purple  solution  if  diluted  with  alcohol  (not  with  water,  which 
destroys  the  colour)  shows  with  a  spectroscope  a  characteristic  ab- 
sorption spectrum  consisting  of  two  absorption  bands,  one  between 
D  and  E  abutting  on  E,  and  a  second  adjoining  the  E  line.  In  the 
earlier  stages  of  the  reaction  a  third  narrow  band  near  D  makes  its 
appearance  but  disappears  later  on.^ 

Pettenkofer' s  reaction  depends  upon  the  presence  in  all  bile- 
acids  of  their  cholalic  acid  constituent.  On  the  first  addition  of 
sulphuric  acid,  if  the  solution  be  at  all  concentrated,  a  white  pre- 
cipitate may  often  be  observed  consisting  of  cholalic  acid ;  this  is 
dissolved  on  the  further  addition  of  acid,  after  which  the  charac- 
teristic colour  makes'  its  appearance.  It  has  also  recently  been 
shown  that  the  reaction  depends    upon    the  formation    of   fur- 

1  Maly  u.  Emich,  Monatshefte  f.  Chem.  Bd.  iv.  (1883),  S.  89.  See  also 
Hammarsten,  Pfliiger's  Arch.  Bd.  iii.  (1870),  S.  53.  On  the  similar  behaviour  of 
taurocholic  acid  to  gelatin  and  its  peptones  see  Emich,  Monatshefte  J".  Chem. 
Bd.  VI.   (1885),  S.  95. 

-  Maly  u.  Emich,  loc.  cit.  See  also  Lindberger  (Swedish).  See  Abstr.  in  ^Maly's 
Jahresh.  1884,  S.  334. 

3  Pettenkofer,  Annal.  d.  Chem.  u.  Pharm.  Bd.  i.ii.  (1844),  S.  90. 

*  To  avoid  this,  Drechsel  recommends  the  employment  of  phosphoric  acid  (5  of 
glacial  acid  to  1  of  water)  instead  of  sulphuric  acid,  Jn.  f.  prakt.  Chem.  Bd.  xxiv. 
(^1881),  S.  44;  xxvii.  (1883),  S.  424.  In  this  ca.se  tlie  solution  must  be  heated  by 
immersion  in  boiling  water. 

*  Schenk.  See  ref.  in  Malv's  Jahresh.  1872,  S.  232.  Udranszky,  Zt.  f.  phjsiol. 
Chem.  Bd.  xn.  (1888),  S.  372.  'Mac  Munn,  Clin.  chem.  of  urine,  1889,  p.  n4. 


214  PETTENKOFER'S   EEACTION. 

furol  ^  by  the  action  of  the  sulphuric  acid  upon  the  sugar, 
the  colour  arising  from  the  interaction  of  furfurol  with  cholalic 
acid.^ 

It  is  important  to  remember  that  an  extended  series  of  sub- 
stances other  than  cholalic  acid  and  the  bile-acids  (pigments  and 
other  substances  which  are  charred  by  sulphuric  acid)  either 
interfere  with  the  brilliancy  of  the  reaction  or  else  themselves 
yield  a  purple  colour  which  closely  resembles  that  due  to  the 
bile-acids.  Among  the  latter  those  of  chief  importance  are  pro- 
teids,  amyl-alcohol,  oleic  acid,  the  higher  fatty  acids,  and  cho- 
lesterin.^  A  further  element  of  uncertainty  is  introduced  by  the 
fact  that  if  the  suspected  solution  be  extremely  dilute  no  charac- 
teristic colour  is  obtained  although  bile-acids  may  be  present. 
All  the  above  militate  against  the  detection  of  bile-acids  in  fluids 
such  as  urine,  in  which  their  determination  is  a  matter  of  not  in- 
frequent importance.  The  application  of  Pettenkofer's  reaction 
in  its  original  form  has  hence  been  modified  in  details  by  many 
observers  with  a  view  to  rendering  it  more  decisive  and  delicate. 
The  decisiveness  of  the  reaction  is  ensured  by  careful  spectro- 
scopic examination  of  the  absorption  spectrum  of  the  coloured 
solution,  since  the  colours  produced  by  the  majority  of  those 
substances  which  yield  a  reaction  resembling  that  produced  by 
cholalic  acid,  show  no  absorption  bands  in  their  spectra.  Some 
few  however  do  exhibit  absorption  bands  which  fortunately  oc- 
cupy a  different  position  in  the  spectrum  from  those  shown  by 
cholalic  acid  (Udranszky).  If  the  suspected  solution  is  ex- 
tremely dilute  it  may  frequently  be  made  to  yield  Pettenkofer's 
reaction  directly  by  a  previous  concentration  on  the  water-bath. 
A  further  modification  which  is  applicable  to  dilute  solutions  is 
the  following.  A  little  cane-sugar  is  dissolved  in  the  solution 
and  a  strip  of  filter-paper  dipped  into  it  and  then  air- dried. 
When  dry  one  drop  of  concentrated  sulphuric  acid  is  applied  to 
the  paper  with  a  glass  rod.  If  bile-salts  are  present  (even  to  the 
extent  of  -03  p.  c.)  a  distinct  violet  stain  may  be  observed  on  the 
paper  after  standing  for  a  quarter  of  a  minute :  the  stain  is  most 
easily  seen  by  transmitted  light.*  Instead  of  sugar  an  aqueous 
(O'l  p.  c.)  solution  of  furfurol  may  be  used  to  great  advantage  as 
follows.  One  drop  of  this  solution  is  added  to  1  c.  c.  of  the  sus- 
pected solution,  either  aqueous  or  alcoholic,  in  a  test  tube.  To 
the  above  is  then  added  1  c.  c.  of  concentrated  sulphuric  acid  and 
the  mixture  is  cooled  under  water  so  that  its  temperature  does 
not  exceed  50°  —  60°  C.  To  detect  bile-acids  in  urine  with  ab- 
solute certainty  it  is  essential  to  separate  them  from  this  excre- 

1  Also  known  as  furfuraldehvde  C4H3O  .  COH,  the  aldehvde  of  pyromucic  acid 
C4H3O.COOH. 

2  Mylius,  Zt.f.  phi/siol.  Chem.  Bd.  xi.  (1887),  S.  492. 

2  For  a  complete  list  of  these  see  Udranszky,  loc.  cit.  S.  358. 
*  Strassburg,  Pfliiger's  Arch.  Bd.  iv.  (187lf,  S.  461. 


CHEMICAL   BASIS   OF  THE   ANIMAL   BODY.        215 

tion  before  applying  Pettenkofer's  test.  This  is  effected  either  by 
precipitation  with  basic  lead  acetate  or  extraction  with  alcohol  or 
chloroform.^ 


THE   COLOUEINa   MATTERS   AND   PIGMENTS 
OF   THE   ANIMAL   BODY. 

HEMOGLOBIN  AND    ITS    DERIVATIVES. 

1.  Haemoglobin.^  This  is  the  well-known  constituent  of  the 
red  corpuscles  to  which  the  dark  colour  of  the  blood  from  an 
asphyxiated  animal  is  due.  It  is  also  present  to  a  less  and 
somewhat  variable  amount  in  ordinary  venous  blood,  in  presence 
of  correspondingly  variable  amounts  of  the  compound  which  it 
forms  with  oxygen,  namely  oxy-hfemoglobin.  In  normal  arterial 
blood  it  is  probably  present  in  mere  traces,  if  at  all,  since  here  its 
affinities  for  oxygen  are  completely  satisfied  to  form  oxy-hsemo- 
globin.  Haemoglobin  is  chiefly  of  interest  as  an  oxygen-carrier  or 
respiratory  pigment,  in  virtue  of  the  ease  with  which  it  absorbs 
and  unites  in  loose  combination  with  oxygen  when  merely  ex- 
posed to  this  gas,  and  again  gives  it  up  when  brought  into  rela- 
tionship with  the  oxygen-free  tissues  of  the  body.  The  conditions 
and  phenomena  of  this  fixation  and  liberation  of  oxygen  by  haemo- 
globin have  been  very  fully  investigated ;  the  fundamentally  im- 
portant facts  in  connection  with  it  have  already  been  stated  in 
some  detail  in  an  earlier  part  of  this  work  (§  343  et  seq.),  so  that 
it  is  now  only  necessary  to  add  some  further  details  of  haemoglobin 
of  a  more  purely  chemical  character. 

Owing  to  the  ease  and  avidity  with  which  hsemoglobin  unites 
with  oxygen  to  form  the  distinct  and  stable  compound  known  as 
oxy-hsemoglobin,  its  investigation  is  attended  with  considerable 
experimental  difficulties ;  hence  our  knowledge  of  it  as  a  chemi- 
cal substance  is  on  the  whole  less  complete  than  is  that  of  oxy- 
hsemoglobin.  Hsemoglobin  may  be  obtained  in  a  crystalline 
form,-^  but  with  some  considerable  difficulty  owing  to  its  extreme 
solubility  in  water.  The  crystals  may  be  prepared  by  sealing  up 
a  concentrated  aqueous  solution  of  oxy-hsemoglobin  in  glass  tubes 
from  which,  if  necessary,  all  remaining  air  is  displaced  by  hydro- 
gen :  on  prolonged  standing  all  the  oxygen  disappears  during  the 
putrefactive  reduction  which  ensues,  and  finally,  more  readily  on 
exposure  to  a  low  temperature,  crystals  of  hsemoglobin  make  their 

1  For  details  see  Hoppe-Seyler,  Hdbch.  d.  phys.-patJt.  Ckem.  Anal.  1883,  S.  399, 
and  Neubauer  u.  Vogel,  Anahjse  d.  Hams,  1890,  S.  146. 

2  The  single  name  hi3emoglobin  is  used  here  to  denote  what  is  more  frequently 
and  usually  called  '  reduced  '  hsemoglobin,  as  distinct  from  oxy-hajmoglobin.  The 
adoption  of  the  name  as  here  used  is  both  simpler  and  more  logical. 

3  First  described  by  Kiihne,  Virchow's  Arch.  Bd.  xxxiv.  (1855),  S.  423. 


216  HEMOGLOBIN. 

appearance  in  the  fluid.^  A  similar  production  and  formation  of 
crystals  is  frequently  observed  when  crystals  of  oxy-hsemoglobin 
are  sealed  up  with  Canada  balsam  under  a  cover-slip  and  kept  for 
some  time.2  The  form  of  the  crystals  obtained  from  the  blood  of 
different  animals  has  not  yet  been  fully  investigated.  They  ex- 
hibit to  a  marked  degree  the  phenomena  of  pleochroism,  being  ap- 
parently trichromatic.^ 

Pleochroism  is  that  property  possessed  by  many  crystals  of  appear- 
ing to  differ  more  or  less  in  colour,  in  accordance  with  the  direction 
from  which  they  are  viewed  by  transmitted  light.  The  phenomena 
are  usually  investigated  by  means  of  a  single  ISTicol  prism.  For 
further  details  consult  some  special  work  on  mineralogy  or  the  article  on 
this  subject  in  the  "Encyclopaedia  Britannica,"  Vol.  xvi.  p.  375. 

As  ordinarily  seen  the  crystals  of  haemoglobin  have  a  dark  red 
appearance,  unlike  the  bright  scarlet  of  oxy-hsemoglobin,  with  a 
strong  purple  or  bluish  tint.  They  are  extremely  soluble  in  water, 
much  more  so  than  the  crystals  of  oxy-haemoglobin.  The  optical 
properties  of  solutions  of  haemoglobin  have  already  been  sufh- 
ciently  described  (§  346,  and  see  below  Fig.  36,  No.  5).  One  of 
the  most  remarkable  properties  of  haemoglobin  is  its  power  of 
uniting  directly  with  any  one  of  several  gases,  such  as  oxygen, 
carbon  monoxide,  nitric  oxide  and,  as  recent  research  has  shown, 
possibly  carbon  dioxide  ;  the  compounds  which  are  thus  formed 
have  in  the  case  of  the  first  three  gases  a  definite  and  constant 
composition,  crystallising  more  or  less  readily  in  characteristic 
forms  and  showing  in  aqueous  solutions  absorption  spectra  which 
are  constant  and  characteristic  for  each.     (See  below.) 

The  chemical  composition  of  haemoglobin  does  not  as  yet  admit 
of  being  represented  by  any  definite  formula,  and  indeed  its  per- 
centage composition  has  not  been  determined  by  direct  analysis. 
It  must  be  inferred  from  a  knowledge  of  the  probable  composi- 
tion of  the  more  stable  and  easily  crystallisable  oxy-haemoglobin 
and  of  the  quantitative  relationships  which  hold  good  between 
haemoglobin  and  oxygen  during  its  conversion  into  oxy-haemo- 
globin. As  will  be  seen  later  on,  analysis  of  purified  crystals  of 
oxy-haemoglobin  shows  that  these  probably  differ  in  composition 
as  prepared  from  the  blood  of  different  animals,  and  the  same 
statement  therefore  probably  holds  good  for  haemoglobin.  When 
decomposed  in  the  absence  of  oxygen  (air),  as  for  instance  by  the 
action  of  organic  acids,  more  dilute  mineral  acids,  or  best  of  all  by 
caustic  alkalis,  it  yields  a  proteid,  of  which  but  little  is  known 
(see  p.  32),  and  a  coloured  substance  called  by  Hoppe-Seyler 
haemochromogen.     The  latter  on  exposure  to  air  absorbs  oxygen 

1  Hiifner,  Zt.  f.  physiol.  Chem.  Bd.  iv.  (1880),  S.  382.  Cf.  Nencki  u.  Sieber, 
Ber.  d.  d.  chem.   GeseU.  Bd.  xix.   (1886),  Sn.  129,  410. 

2  A.  Ewald,  Zt.f.  Biol.  Bd.  xxir.  (1886),  S.  459. 

3  A.  Ewald,  loc.  cit. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        217 

and  becomes  ordinary  lisematin ;  it  is  in  fact  the  substance  usually- 
spoken  of  as  reduced  hsematin.     (See  below.) 

2.  Oxy-hsBinoglobin.  When  haemoglobin  is  exposed  to  the 
air  it  rapidly  unites,  molecule  for  molecule,  with  oxygen,  thus 
becoming  oxy-hsemoglobin,  the  characteristic  constituent  of  the 
red-corpuscles  to  which  the  scarlet  colour  of  arterial  blood  is  due.^ 
It  may  be  readily  set  free  from  the  corpuscles  by  the  addition  to 
defibrinated  blood  of  such  fluids  as  alcohol,  ether,  chloroform, 
water,  and  solutions  of  bile-salts,  or  by  repeatedly  freezing  and 
thawing  the  blood;  when  thus  set  free  it  passes  into  solution  in 
the  adjacent  serum.  Erom  this  solution  it  may  be  obtained  as 
crystals  with  more  or  less  readiness,  dependently  upon  the  kind 
of  animal  whose  blood  is  used  for  its  preparation  (see  §  344),  the 
difference  being  due,  partly  at  least,  to  the  varying  solubility  of 
the  several  hsemoglobins. 

To  obtain  rapidly  a  microscopic  preparation  of  oxy-hsemoglobin 
crystals  it  suffices  to  take  a  drop  of  the  blood  of  some  animal 
whose  hsemoglobin  crystallises  readily  (rat,  guinea-pig,  or  dog),  to 
mix  a  drop  of  it  on  a  slide  with  a  minute  drop  of  water,  and  allow 
the  mixture  to  evaporate  until  a  ring  of  dried  substance  is  formed 
at  the  periphery.  If  it  be  now  covered  with  a  cover-slip,  crystals 
usually  form  in  a  short  time,  especially  if  it  be  kept  cooled.  For 
the  preparation  of  oxy-hsemoglobin  crystals  on  a  large  scale  many 
methods,  the  same  in  general  principles  but  differing  somewhat 
in  detail,  have  been  proposed,  the  difficulty  of  the  preparation 
varying  considerably  according  to  the  kind  of  blood  used.^  For 
laboratory  purposes  large  quantities  of  crystallised  oxy-ha^mo- 
globin  may  be  very  readily  obtained  from  dog's  blood  as  follows 
(Kuhne).  The  blood  is  defibrinated  and  strained  through  fine 
muslin:  it  is  then  placed  in  a  flask  and  ether  is  added  with 
frequent  shaking  until  the  blood  is  just  '  laky,'  i.  e.  transparent. 
The  flask  is  now  surrounded  by  a  freezing  mixture  of  ice  and  salt 
and  in  a  short  time  its  contents  usually  become  almost  pasty 
from  the  mass  of  crystals  which  form  in  it.  These  are  then 
centrifugalised  off,  dissolved  in  a  minimal  amount  of  water, 
filtered,  cooled  to  0°,  and  after  the  addition  of  one  quarter  of  its 
bulk  of  cooled  alcohol  again  immersed  in  a  freezing  mixture. 
The  second  crop  of  crystals  thus  obtained  may  be  again  recrys- 
tallised  as  already  described.  The  crystals  are  finally  washed 
with  water  at  0°  containing  25  p.  c.  of  alcohol,  and  may  be  dried 
in  vacuo  over  sulphuric  acid  at  0°,  and  are  now  fairly  stable. 

1  Hsemoglobin  is  united  to  corpuscles  in  the  blood  of  all  vertebrates,  with  two 
exceptions.  In  invertebrate  blood  it  is  usually  found  in  solution  in  the  plasma, 
but  there  are  a  few  (eif^ht)  exceptions  to  this  rule.  For  details  and  literature  see 
Halliburton,  Chem.  Phi/siol.  and  Pathol.  1891,  pp.  267,  316. 

2  For  fuller  details  see  Gamgee,  Physiol.  Chemistry,  Vol.  i.  1880,  p.  85.  See 
later  Otto,  Zt.  f.  physiol.  Chem.  Bd.  vii.  (1882),  S.  57.  Zinoffsky,  Ibid.  Bd.  x. 
(1885),  S.  18.  Hiifner,  Beitr.  z.  Physiol.  Festschr.  f.  C.  Ludwixj,  1887,  S.  74. 
Mavet,  Comft.  Rend.  T.  109  (1890),  p'.  156. 


218  OXY-H^MOGLOBIN. 

The  crystals  obtained  from  the  heemoglobin  of  various  animals 
differ  in  their  crystalline  form.  The  following  figure  shows  some 
of  the  most  typical  forms. ^ 


Fig.  35.     Crystals  of  Oxy-H/Emoglobin.     (After  Funke.) 
a.  Squirrel,     b.  Guinea-pig,     c.  Cat,  or  Dog,     d.  Man,     e.  Hamster. 

Apart  from  these  differences  in  crystalline  form  the  oxy-hsemo- 
globin  of  different  animals  varies  in  its  solubility,  in  the  amount 
of  water  of  crystallisation  with  which  its  crystals  are  united,  and 
also  apparently  in  its  percentage  composition.  The  crystals  are 
pleochroic  but  to  a  less  extent  than  are  those  of  hsfimoglobin.^ 
As  against  these  differences  it  is  important  to  notice  that  the 
close  relationship  of  the  various  forms  of  oxy-hsemoglobin,  from 
whatever  blood  they  may  be  obtained,  is  shown  by  the  fact  that 
the  spectroscopic  properties  are  in  all  cases  identical,  as  also  are 
the  products  of  decomposition  and  the  compounds  formed  with 
gases.  Numerous  analyses  of  oxy-hsemoglobin  have  been  made,^ 
but  these  while  they  tell  us  at  most  that  it  consists  of  oxygen, 
hydrogen,  nitrogen,  and  carbon  together  with  iron  as  a  character- 
istic constituent  and  some  sulphur,  and  seem  to  indicate  that  it 
differs  in  composition  as  obtained  from  different  animals,  do  not 
as  yet  enable  us  to  assign  with  any  certainty  a  definite  formula 
to  its  composition.  It  is  however  certain  that  its  molecular 
weight  is  enormously  great   (13,000  — 14,000).^ 

1  For  a  discussion  of  the  various  crystalline  forms  of  oxy-liEemoglobin  see 
Halliburton,  Chem.  Physiol,  and  Pathol.  1891,  p.  270. 

2  A.  Ewald,  loc.  cit.  (sub  hsemoglobin). 

*  See  Hammarsten's  Lehrb.  d.  phi/siol.  Chem.  1891,  S.  57;  or  Halliburton's 
Text-book  of  Chem.  Physiol.  Pathol.  1891,  p.  286. 

*  Marshall,  Zf.  f.  physiol.  Chem.  Bd.  vn.  (1882),  S.  81.  Kiilz,  Ibid.  S.  384. 
Cf.  Zinoffsky,  Ibid.  Bd.  x.  (1886),  S.  16,  and  .see  Hufner,  loc.  cit. 


CHEMICAL  BASIS   OF  THE   ANIMAL   BODY, 


219 


CO 


M 


^  iffl  (XI 

Fig.  36.  (After  Freyer  and  Gamgee.)  The  Spectra  of  Oxy-h.^moglobin  in 
different  grades  of  concentration,  of  (reduced)  hemoglobin,  and  of 
Carbon-Monoxide-Hemoglobin. 

1  to  4.     Solution  of  Oxj'-hsemoglobin  containing  (1)  less  than  'Ol  p.c,  (2)  '09  p.c, 
(3)  -37  p.c,  (4)  '8  p.c. 

5.  "      "     (reduced)  Haemoglobin  containing  about  -2  p.c. 

6.  "      "     carbon-monoxide  Hemoglobin. 

In  each  of  the  six  cases  the  layer  brought  before  the  sjiectroscope  was  1  cm.  in 
thickness.  The  letters  (A,  a,  &c.)  indicate  Frauenhofer's  lines,  and  the  figures  wave- 
lengths expressed  in  100,000t]i  of  a  millimeter. 


220  OXY-H^MOGLOBIN". 

The  spectroscopic  appearances  of  solutions  of  oxy-hsemoglobin 
have  been  already  sufficiently  described  and  figured  (§  345).  (For 
convenience  of  reference  Fig.  75  is  reproduced  here.)  When  its 
solutions  are  heated  or  it  is  treated  either  in  solution  or  as  a  solid 
with  acids  or  alkalis,  it  may  be  readily  decomposed,  yielding  a 
proteid  as  in  the  case  of  haemoglobin  and  a  coloured  residue,  viz. 
hsematin.  (See  below.)  The  oxygen  which  is  loosely  combined 
with  hsemoglobin  in  the  formation  of  oxy-hsemoglobin  may  be 
readily  removed  by  several  means  of  which  the  following  are 
those  most  usually  employed. 

(i)  The  solution  is  warmed  to  40°  and  the  gas  driven  off  by 
exposure  to  the  vacuum  of  a  mercurial  pump,  (ii)  A  current  of 
some  neutral  gas  such  as  hydrogen  or  nitrogen  is  passed  through 
the  solution,  (iii)  The  solution  is  treated  with  a  few  drops  of 
some  reducing  agent  such  as  Stokes'  fluid.^  This  is  prepared  by 
adding  tartaric  or  citric  acid  to  a  solution  of  ferrous  sulphate,  and 
then  ammonia  until  it  is  strongly  alkaline.  This  reagent  does  not 
keep  and  must  be  freshly  prepared  each  time  it  is  required.  In- 
stead of  Stokes'  fluid,  ammonium  sulphide  may  be  used,  but  in 
this  case  some  slight  manipulation  is  frequently  required  to  ensure 
reduction.  A  few  drops  of  the  sulphide  are  added  to  the  solution, 
which  is  then  gently  warmed :  if  on  examination  with  the  spectro- 
scope it  is  found  that  the  reduction  has  not  taken  place,  as  shown 
by  the  persistence  of  the  two  bands  of  oxy-hsemoglobin,  a  little 
more  of  the  sulphide  may  be  added  and  the  mixture  again  care- 
fully warmed. 

The  amount  of  oxygen,  removable  by  the  means  just  described, 
with  which  one  gram  of  haemoglobin  (from  dog's  blood)  can  unite 
is  usually  stated  as  being  1'59  c.c.  at  0°  and  760  mm.  Hg.  this 
constant  being  taken  as  independent  of  the  concentration  of  the 
solutions  employed.^  Quite  recently  some  doubt  has  been  cast  on 
the  quantity  being  thus  constant ;  and  it  has  been  stated  that 
several  modifications  of  hsemoglobin  exist  which,  while  they  can- 
not be  discriminated  by  their  purely  chemical  characteristics, 
exhibit  a  marked  difference  as  to  the  amount  of  oxygen  with 
which  the  same  quantity  of  each  can  unite  under  similar  external 
conditions ;  the  results  thus  obtained  are  stated  to  hold  good  for 
the  compound  of  oxygen  with  hsemoglobin  as  it  exists  in  the  red 
blood-corpuscles  of  the  dog,^  and  further  for  the  hsemoglobin  of 
guinea-pigs  and  geese.*  Further  investigation  must  decide  the 
interesting  questions  raised  by  the  above  statements. 

There  appears  to  be  a  consensus  of  opinion  that  hsemoglobin, 
and   more    particularly    oxy-hsemoglobin,   possesses   to   a   slight 

1  Proc.  Roy.  Soc.  June,  1864.     Phil.  Mag.   November,  1864. 

2  Hiifner,  Zt.  f.  pkysiol.  Chem.  Bd.  i.  (1878),  Sn.  317,  386.  See  also  Jn.  f. 
prakt.  Chem.  Bd.  xxii.  (1880),  S.  362. 

3  Bohr  u.  Torup,  Skandinav.  Arch.  f.  Physiol.  Bd.  iii.  Hft.  1,  2  (1891),  S.  69. 
Bohr, /6(W.  Sn.  76,  101. 

4  Jolin,  Arch.f.  Physiol.  Jahrg.  1889,  S.  265. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        221 

degree  the  properties  of  an  acid.  This  view  appears  to  be  based 
on  the  following  facts.  Oxy-hsemoglobin  is  extraordinarily  soluble 
in  alkalis  and  in  this  solution  appears  to  be  more  stable  than 
ordinarily.  It  is  further  stated  that  it  has  a  feeble  power  of 
facilitating  the  evolution  of  carbon-dioxide  from  dilute  solutions 
of  sodium  carbonate.^  It  is  hence  often  supposed  that  in  the  red 
.  blood-corpuscles  the  haemoglobin  is  united  to  the  alkalis  of  which 
their  stroma  partially  consists.  If  the  above  views  are  correct 
they  may  assist  in  explaining  to  some  slight  extent  the  difficulties 
in  understanding  the  causes  of  the  exit  of  carbon-dioxide  from 
venous  blood  during  its  passage  through  the  lungs.  (See  §  357.) 
But  the  possibility  here  indicated  must  be  received  with  the 
greatest  caution ;  for  it  has  been  shown  that  although  a  dilute 
alkaline  solution  of  oxy-hsemoglobin  when  exposed  to  a  low 
partial  pressure  of  carbon-dioxide  absorbs  less  of  this  gas  than 
suffices  to  convert  the  alkali  into  bicarbonate,  thus  acting  like 
an  acid,  at  higher  partial  pressures  it  absorbs  more  than  can 
be  accounted  for  by  the  change  of  the  alkali  into  bicarbonate. 
In  the  latter  case  the  haemoglobin  seems  to  act  like  a  feeble 
base.2  It  is  interesting  here  to  notice  that  if  the  immediately 
preceding  statements  hold  good,  the  hcemogiobin  must  possess 
increasingly  acid  properties  in  proportion  as  the  carbon-dioxide 
begins  to  l3e  evolved  from  the  blood,  and  might  thus  further 
that  exit.  The  power  apparently  possessed  by  haemoglobin  of 
itself  uniting  directly  with  carbon-dioxide  will  be  referred  to 
again  later  on. 

3.  Carbon-monoxide  haemoglobin.  When  a  current  of  car- 
bon-monoxide is  passed  through  a  solution  of  oxy-hsemoglobin  the 
oxygen  is  driven  off  and  its  place  taken  by  the  first-named  gas. 
The  compound  thus  formed  results,  like  oxy-hsemoglobin,  from  the 
union  of  one  molecule  of  the  gas  with  one  of  haemoglobin.  It 
further  resembles  oxy-hsemoglobin  in  being  readily  crystallisable  ^ 
in  forms  isomorphous  with  those  of  the  former,  but  the  crystals 
are  on  the  whole  less  soluble,  brighter  coloured  and  more  stable 
than  are  those  of  oxy-haemoglobin.*  They  are  distinctly  dichro- 
matic (see  p.  216).  The  compound  of  carbon-monoxide  with 
haemoglobin  is  much  more  stable  than  is  oxy -haemoglobin,  so  that 
the  gas  is  not  again  expelled  by  the  action  of  oxygen,  a  fact  which 
fully  explains  the  fatal  result  of  breathing  carbon-monoxide. 
Finally  the  spectrum  of  carbon-monoxide  haemoglobin  while  very 
similar  at  first  sight  to  that  of  oxy-haemoglobin,  differs  distinctly 

1  Preyer,  Die  Blutkry stalk,  1871,  S.  70. 

'-2  Setschenow,  Mem.  de  I'acad.  de  St.  Petersh.  T.  xxvi.  1879,  confirmed  by 
Zuntz,  Hermann's  Hdhch.  d.  Physiol.  Bd.  iv.  Th.  2  (1882),  S.  76. 

3  For  preparation  in  quantity  see  Kiilz,  Zt.  f.  physiol.  Chem.  Bd.  vii.  (1882), 
S.  385. 

*  Carbon-monoxide  lisemoglobin  is  unaffected  by  either  putrefactive  changes  or 
the  action  of  pancreatic  juice.     Hoppe-Seyler,  Ibid.  Bd.  i.  (1877),  S.  131. 


222  CAEBON-DIOXIDE   HEMOGLOBIN. 

from  it  in  the  position  of  its  two  absorption  bands  (see  Fig.  36, 
iSTo.  6).  The  spectrum  of  this  compound  undergoes  no  change  by 
the  action  of  any  of  the  reducing  agents  described  on  p.  220  : 
this  affords  a  further  characteristic  means  of  discriminating  be- 
tween the  compounds  of  carbon-monoxide  and  oxygen  with 
hsmoclobin.  Since  the  determination  of  this  compound  in  blood 
is  frequently  of  considerable  importance  in  medical  jurisprudence, 
many  tests  for  its  presence  have  been  devised  additionally  to  the 
evidence  afforded  by  the  spectroscope.  One  of  the  oldest  and 
best  is  due  to  Hoppe-Seyler.i  It  consists  in  adding  to  the  sus- 
pected blood  twice  its  volume  of  caustic  soda  of  sp.  gr.  1-3.  If 
carbon-monoxide  hsemoglobin  is  present  it  yields  a  brilliant  red 
precipitate,  differing  entirely  in  appearance  from  the  brownish- 
green  mass  observed  if  oxy-heemoglobin  is  present.  For  further 
tests  consult  the  literature  quoted  below.^ 

4.  Nitric  oxide  haemoglobin.  If  a  current  of  nitric  oxide 
be  passed  through  a  solution  of  carbon-monoxide  haemoglobin,  the 
carbon-monoxide  is  displaced  by  the  former  gas.^  The  compound 
thus  obtained  is  still  more  stable  than  is  carbon-monoxide  hsemo- 
globin.  It  may  be  crystallised  and  in  solution  exhibits  two 
absorption  bands  very  similar  to  those  of  oxy-heemoglobin  but 
slightly  nearer  the  red  end  of  the  spectrum ;  these  bands  are  not 
affected  by  reducing  agents.  If  prepared  by  passing  the  gas 
through  ordinary  blood,  the  latter  should  first  be  freed  from 
oxygen  by  a  current  of  hydrogen  and  care  must  be  taken  to 
neutralise  the  nitrous  acid  formed  during  the  process. 

5.  Carbon-dioxide  haemoglobin.  The  possible  union  of 
carbon-dioxide  with  haemoglobin  has  already  been  referred  to 
(p.  221),  and  more  recent  researches  have  thrown  further,  though 
still  far  from  complete  light  upon  this  possibility.  There  appears 
to  be  no  doubt  that  a  solution  of  hsemoglobin  takes  up  a  much 
larger  volume  of  carbon-dioxide  than  can  be  accounted  for  as  the 
result  of  a  merely  physical  absorption.  Thus  in  one  set  of  experi- 
ments it  was  found*  that  1  gr.  of  haemoglobin  could  unite  with 
2-366  c.c.  of  the  gas  at  a  temperature  of  184°  and  partial  pressure 
of  31-98  mm.  of  Hg,  the  latter  being  a  mean  average  partial 
pressure  of  carbon-dioxide  in  venous  blood  according  to  the  older 

1  Virchow's  Arch.  Bd.  xiii.  (1858),  S.  104.  For  a  recent  modification  of  this 
test  see  E.  Salkowski,  Zt.  f.  physioL.  Chem.  Bd.  xii.  (1888),  S.  227. 

^  Jaderholm  (Swedish),'  Abst.  in  Maly's  Jahresb.  1874,  S.  102.  Weyl  u.  von 
Anrep,  Arch.  f.  Physiol.  Jahrg.  1880,  S.  227.  Zaleski,  Zt.  f.  phjiswl.  Chem.  Bd.  ix. 
(1885),  S.  225.  Kunkel,  Sitzb.  d.  Wiirzb.  physik.-m.ed.  Gesell.  1888,  Sitz.  9. 
Katayama,  Virchow's  Arch.  Bd.  cxiv.  (1889),  S.  53.  Welzel,  Verhandl.  d.  physik.- 
med.  Gesell.  Wiirzb.  (N.  F. )  Bd.  xxiii.  (1889),  S.  3. 

^  L.  Hermann,  Arch.f.  Anat.  u.  Physiol.  Jahrg.  1865,  S.  409. 

*  Bohr,  see  Beitrdqe  z.  Physiol.  Ludwig,  gewidmet,  1887,  S.  164.  Centralb.  f. 
Physiol.  Bd.  IV.  (1890).  S.  253.  Skandinav.  Arch.  f.  Physiol.  Bd.  ill.  Hf.  1,  2 
(1891),  S.  47.     See  also  Jolin,  Arch.f.  Physiol.  Jahrg.  1889,  Sn.  277,  285. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.       223 

established  data/  while  that  in  arterial  blood  is  21  "28  mm.^  It 
is  further  stated  that  the  stronger  solutions  of  haemoglobin  absorb 
relatively  less  carbon-dioxide  than  the  weaker,  and  that,  as  in  the 
case  of  oxy-hsemoglobin  (see  p.  221)  various  modifications  of 
haemoglobin  exist  possessing  difi'erent  powers  of  uniting  with  this 
gas.  On  comparing  the  amounts  of  carbon-dioxide  and  of  oxygen 
or  CO  or  NO  which  may  unite  with  a  given  weight  of  haemoglobin 
it  is  at  once  evident  that  the  mode  of  union  of  the  former  gas 
must  be  different  from  that  of  the  latter  three,  with  which,  as 
already  stated,  haemoglobin  unites  molecule  for  molecule.  This 
difference  in  behaviour  is  very  probably  due  to  the  decomposition 
which  haemoglobin  undergoes  when  a  current  of  carbon-dioxide  is 
passed  through  it,^  and  indeed  it  is  hence  probable  that  the  so- 
called  carbon-dioxide  haemoglobin  is  rather  a  compound  of  the  gas 
with  a  coloured  product  of  the  decomposition  of  haemoglobin,  viz. 
haemochromogen,  which  has  been  shown  by  Hoppe-Seyler  to  unite 
with  carbon-monoxide  (see  below).  The  compound,  whatever  be 
its  true  nature,  is  stated  to  exhibit  a  one-banded  absorption  spec- 
trum closely  similar  to  that  of  haemoglobin,  but  the  centre  of  the 
band  lies  slightly  more  towards  the  violet  end  of  the  spectrum.* 
Bohr  states  that  the  absorption  of  carbon-dioxide  is  independent 
of  the  simultaneous  presence  of  oxygen.^ 

The  accurate  quantitative  determination  of  the  amount  of  haemo- 
globin in  any  given  solution  is  a  matter  of  extreme  importance,  not 
merely  in  connection  with  several  of  the  statements  contained  in  the 
preceding  description  of  haemoglobin  and  its  compounds  with  gases, 
but  also  in  many  investigations  which  turn  on  tlie  varying  amounts 
of  this  substance  under  different  experimental  conditions,  and  further 
for  clinical  purposes.  It  may  therefore  not  be  out  of  place  to  describe 
briefly  the  principles  on  which  the  determinations  are  based,  referring 
the  reader  to  special  works  for  the  details  of  the  respective  processes. 

The  methods  employed  fall  under  two  categories:  chemical  and 
physical. 

1.  Chemical,  a.  The  amount  of  iron  present  in  100  parts  of 
haemoglobin  has  been  frequently  determined  for  the  blood  of  various 
animals.  It  may  be  stated  to  be  about  -43 — -45  p.c.  Hence  if  a 
solution  of  this  substance  be  evaporated  to  dryness  and  the  residue 
incinerated,   the  amount  of   haemoglobin  may   be   inferred  from  the 

1  See  Wolffberg,  Pfluger's  Arch.  Bd.  vi.  (1872),  S.  23.  Strassburg,  Ibid.  S.  65 
Nussbaum,  Unci.  Bd.  vii.  (1873),  S.  296. 

■-i  But  cf.  Bohr,  Centralb.  J.  Phi/swl.  Bd.  i.  (1887),  S.  293,  ii.  (1888),  S.  437,  who 
makes  it  much  less.  According  to  this  observer  the  partial  pressure  of  COo  in 
blood  is  less  than  that  of  expired  air,  and  that  of  oxygen  is  greater.  If  this  should 
prove  to  be  the  case  on  further  investigation,  it  would  appear  that  the  gaseous 
interchange  which  takes  place  in  the  lungs  cannot  be  the  result  of  a  purely  diffusive 
process,  as  it  is  now  held  to  be  (§  354 — 357). 

3  Torup  (Swedish).     See  Abst.  in  Maly's  Jahresb.  1887,  S.  115. 

*  Torup,  loc.  cit.  and  see  also  "Ueber  die  Kohlensaurebindung  des  Blutes," 
Kopenhagen,  1887. 

5  Skandinav.  Arck.f.  Physiol  Bd.  in.  (1891),  S.  62. 


224  SPECTEOPHOTOMETKY. 

amount  of  iron,  existing  as  oxide  of  iron,  in  this  residue.^  b.  Since 
tlie  volume  of  oxygen  which  unites  with  a  given  quantity  of  htiemo- 
globin  is  known  witli  considerable  accuracy  (but  see  above,  p.  221), 
the  amount  of  this  substance  may  be  determined  by  saturating  its 
solution  with  oxygen  and  then  estimating  the  volume  of  the  gas 
which  is  united  to  the  hfemoglobin.  The  determination  is  made 
either  by  extracting  the  oxygen  with  a  mercurial  pump  or  displacing 
it  by  carbon-monoxide,  or  estimating  it  in  the  solution  by  a  volu- 
metric process  with  sodium  sulphite  and  indigo.^  These  methods  are 
inferior  to  the  following. 

2.  Physical.  These  may  be  again  divided  into  two :  colorimetric 
and  spectrophotometric. 

(i)  Colorimetric  method.  The  principle  of  this  method  may  be 
briefly  stated  as  follows.  A  standard  solution  of  hsemoglobin  is  pre- 
pared from  pure  crystals  of  the  substance.  The  tint  of  the  solution 
in  which  the  haemoglobin  is  to  be  determined  is  then  compared  with 
that  of  the  standard  solution :  if  it  is  not  the  same  when  examined 
under  the  same  conditions,  it  must  be  equalised  by  either  of  the 
methods  to  be  next  described ;  and  from  the  operations  necessary  to 
produce  equality  of  tint  the  relative  concentrations  of  the  two  solu- 
tions may  be  inferred,  and  hence  the  absolute  concentration  of  the 
unknown  solution.  The  methods  more  usually  employed  consist 
either  in  diluting  one  or  the  other  of  the  solutions  until  their  tint 
is  the  same  when  examined  in  layers  of  equal  thickness  (Hoppe- 
Seyler),^  or  else  in  determining  the  different  thicknesses  of  the  fluid 
layer  of  each  which  exhibits  the  same  tint  (Duboscq).  Since  in  the 
latter  case  the  concentrations  of  the  two  solutions  are  inversely  pro- 
portional to  the  thicknesses  of  their  layers  when  their  tint  is  the 
same,  the  amount  of  haemoglobin  in  the  solution  of  unknown  strength 
can  be  at  once  inferred.''  For  clinical  purposes  Gower's  haemo- 
globinometer  is  perhaps  most  frequently  employed.  In  this  instru- 
ment a  measured  volume  of  blood  is  diluted  till  it  has  the  same  tint 
as  that  of  a  standard  mass  of  gelatin  coloured  with  carmine  and 
picrocarmine.^  There  are,  however,  many  other  forms  of  colorimeter 
designed  for  clinical  use. 

(ii)  Spectrophotometric  method.  All  coloured  substances  in  solu- 
tion possess  the  power  of  absorbing  light.  With  a  given  thickness 
of  a  given  substance  the  amount  of  light  transmitted  by  the  solution 
bears  to  the  incident  light  a  ratio  which,  while  it  varies  for  different 
parts  of  the  spectrum,  is  constant  for  any  one  portion,  and  is  there- 
fore characteristic  of  each  substance.     Hence  if  the  absolute  absorb- 

1  Pelouze,  Compt.  Rend.  T.  i.    (1865),  p.  880. 

'^  Grehant,  Compt.  Rend.  T.  lxxv.  (1872),  p.  495.  Quiuquaud,  Ibid.  T.  lxxvi. 
p.  1489.     Schiitzenberger  et  Risler,  Ibid.  p.  440. 

a  Hdbch.  d.  phi/sioL  pathol.-chem.  Anal.  Aufl.  5  (188-3),  S.  435 

*  For  a  description  of  Duboscq's  and  other  apparatus  see  G.  u.  H.  Kriiss, 
Kolorimetrie  u.  quant.  Spektralanal.  1891,  S.  7  et  seq.  This  work  gives  a_  most 
excellent  account  of  the  best  physical  methods  employed  for  the  determination  of 
colouring  substances  in  solution.  A  useful  review  of  methods  up  to  date  (1882)  is 
given  by  Lambling,  "  Des  proce'des  de  dosage  de  I'he'moglobine,"  Nancy,  1882.  Cf. 
later,  E.  von  Fleischl,  Med.  Jahrb.  1885,  S.  ^425.  Malassez,  Arch.  d.  Phijsiol.  1886, 
p.  257. 

s  For  details  see  Gamgee,  Phjsiol.  Chem.  Vol.  i.  p.  184. 


CHEMICAL   BASIS   OF  THE  ANIMAL   BODY.        225 

ing  power  of  a  given  thickness  of  the  substance  is  determined  once 
for  all  for  a  given  region  of  the  spectrum  under  given  conditions,  it 
becomes  possible  to  determine  the  amount  of  that  substance  in  any 
solution  of  unknown  concentration  by  examining  the  solution  under 
the  same  conditions  in  the  same  part  of  the  spectrum  and  ascertaining 
how  much  light  it  has  absorbed.  Let  /be  the  intensity  of  the  inci- 
dent light,  and  I'  its  reduced  intensity  after  passing  through  m  layers 
of  a  coloured  solution,  each  of  which  reduces  the  initial  intensity  by 

,  then  it  follows  that  /'  =: 

n  ^m 

This  is  true  whatever  be  the  intensity  of  the  incident  ray;  hence  this 
intensity  may  be  taken  =  1,  and  we  have  J'  =  . 

Again,  let  E  denote  the  reciprocal  of  the  number  which  represents 
in  centimeters  that  thickness  of  layer  of  the  absorbing  solution  which 
reduces  the  intensity  of  the  incident  ray  to  -^^  of  its  initial  intensity 
during  its  passage  through  this  layer.^  Then  if  the  solution  be  exam- 
ined in  a  layer  which  is  always  1  cm.  thick,  this  layer  may  be  regarded 

as  made  up  of  E  layers,  each  of  thickness  -^  cm.       Hence   if  in  the 

formula  previously  given  we  put  n  =  10  and  m  —  E,  we  find  that 
the  residual  intensity  1'  of  light  after  passing  through  a  layer  1  cm. 

thick  is  ^'  =  IF-  ^  ^^"^' 

whence  E= — log.  /'. 

It  can  also  be  proved  that  E,  the  coefficient  of  extinction,  is 
directly  proportional  to  the  amount  of  colouring  matter  present  in 
the  solution,  or  in  other  words,  to  its  '  concentration ; '  ^  whence  if  the 
concentration  be  represented  by  C, 

(J 

-j^  =  some  constant  ^  A,  or  C  =  AE. 

This  constant  A  having  been  determined  once  for  all  for  a  given 
substance  in  a  solution  of  known  concentration  and  for  a  given  region 
of  the  spectrum,  the  concentration  of  anj^  solution  of  the  same  sub- 
stance of  unknown  strength  is  obtained  by  simply  multiplying  A.  by 
the  coefficient  of  extinction  E.^ 

Spectrophotometers  are  instruments  by  which  the  value  of  I'  (see 
above)  and  hence  of  E  may  be  determined.     Those  of  Vierordt  ^  and 

^  E  \s  called  '  coefficient  of  extinction,'  a  term  introduced  by  Bunsen  and  Roscoe, 
Pogg.  Annal.  Bd.  ci.  (1857),  S.  235. 

2  The  '  concentration  '  is  the  number  of  grams  of  colouring  substance  dissolved 
in  1  c.c.  of  fluid  (Vierordt). 

3  Called  the  '  absorption  ratio '  by  Vierordt. 

*  The  introduction  of  the  spectrophotometric  method  in  a  reliable  form  is  due 
to  Vierordt,  based  upon  the  photochemical  researches  of  Bunsen  and  Roscoe.  See 
Vierordt,  (i)  "  Anwend.  d.  Spectralapparats  zur  Photometrie  d.  Absorptious-spectren 
u.  z.  quant,  chem.  Anal.,"  Tubingen,  1873,  and  (ii)  "Die  quant.  Spectralanal.  in 
ihrer  Anwend.  auf.  Physiol,  u.  s.  w.,"  Tiibingen,  1876. 

^  loc.  cit.  (i),  S.  52. 

15 


226  METH^MOGLOBIK 

Htifner  ^  have  been  most  generally  used  for  physiological  purposes, 
but  there  are  many  other  forms.^  The  value  of  A  has  been  deter- 
mined by  several  observers  for  haemoglobin,  ^  oxy-haemoglobin,^  carbon- 
monoxide  haemoglobin  ^  and  methaemoglobin,^  for  certain  fixed  parts 
of  the  spectrum;  as  also  its  value  for  bile  and  urinary  pigments. '^  If 
the  value  of  A  has  been  determined  for  two  substances  in  tioo  differ- 
ent parts  of  the  spectrum,  the  amount  of  each  substance  in  a  mixture 
of  the  two  may  be  determined  spectrophotometrically.^  This  is  a 
possibility  of  considerable  importance  when  working  with  blood  in 
which  varying  amounts  of  haemoglobin  and  oxy-hasmoglobin  may 
occur  simultaneously. 

6.  Methsemoglobin.  When  blood  or  solutions  of  lisemo- 
globin  which  have  been  exposed  to  the  air  for  some  time  are 
examined  with  the  spectroscope  they  are  frequently  found  to 
exhibit,  in  addition  to  the  more  or  less  persistent  absorption 
bands  of  oxy-hsemoglobin,  a  marked  band  of  absorption  between 
C  and  D,  closely  resembling  but  differing  slightly  in  position 
from  the  band  which  hsematin  shows  in  acid  solution  (see  below). 
This  band  may  also  frequently  be  observed  in  many  pathological 
fluids,  such  as  those  from  ovarial  cysts,  etc.,  which  are  coloured 
by  blood,  and  in  urine  when  similarly  coloured.^  The  substance 
to  which  the  band  is  due  is  known  as  methsemoglobin. ^^  It  may 
be  readily  prepared  in  the  laboratory  by  the  action  of  many 
reagents  such  as  acids  or  alkalis,  or  more  conveniently  of  certain 
salts,  on  solutions  of  oxy-hsemoglobin.  Of  these  salts  those  which 
may  perhaps  on  the  whole  be  most  advantageously  employed  to 
obtain  the  spectrum  of  methsemoglobin  are  nitrites,^^  potassium 
ferricyanide,  or  potassium  permanganate.^^  With  the  two  latter 
salts  the  spectrum  of  methsemoglobin  may  be  obtained  as  follows. 
To  10  c.c.  of  a  moderately  strong  solution  of  oxy-hsemoglobin 
add  a  few  drops  of  a  dilute  ('5 — I'O  p.c.)  solution  of  either  of  the 
salts  and  warm  very  gently.  If  on  examination  with  a  spectro- 
scope the  two  bands  of  oxy-hsemoglobin  are  still  strongly  visible, 

1  .In.  f.  prakt.  Chem.  N.F.  Bd.  xvi.  (1877),  S.  290.  Cf.  Otto,  Pfluger's  Arch. 
Bd.  XXXVI.  (1885),  S.  12.  Glazebrook  has  constructed  a  modification  of  Hiifner's 
instrument.     See  Lea,  Jl.  of  Physiol.  Vol.  v.   (1883),  p.  2.39. 

^  For  all  details  of  instruments  and  spectrophotometry  in  general  see  G,  u.  H. 
Kriiss,  Kolorim.  u.  quant.  Spektralanal.  1891.  Very  complete  details  are  given  in 
iNTeubauer  u.  Vogel,  Ajialt/se  d.  Hams,  1891,  S.  411. 

3  Hiifner,  Zt.f.  phi/siol.  Chem.  Bd.  iii.  (1879),  S.  7. 

4  Htifner,  Ibid.  Bde.  i.  (1878),  S.  317,  iii.  (1879),  S.  4.  Von  Noorden,  Ibid. 
Bd.  IV.  S.  9.  Otto,  Ibid.  Bd.  vir.  S.  62.  Pfluger's  Arch.  Bd  xxxi.  (1883),  S.  244. 
XXXVI.  (1885),  S.  12.     Sczelkow,  Ibid.  Bd.  xli.  (1887),  S.  373. 

5  Marshall,  Zt.  f.  physio/.  Chem.  Bd.  vii.  (1882),  S.  81. 

6  Otto,  Pfluger's  Arch.  Bd.  xxxi.  (1883),  S.  263. 
1  See  Vierordt,  loc.  cit.  or  G.  u.  H.  Kriiss,  loc.  cit. 
^  Vierordt,  loc.  cit. 

9  Hoppe-Seyler,  Zt.  f.  physioL  Chem.  Bd.  v.  (1881),  S.  6. 

10  The  name  was  first  used  by  Hoppe-Seyler  in  1865,  Centralb.  f.  d.  me.d.  Wiss. 
S.  65.  But  see  also  previously  Ibid.  1864,  S.  834,  and  Virchow's  Arch.  Bd.  xxix. 
(1864),  Sn.  233,  u.  597. 

11  Gamgee,  Phil.  Trans.  1868,  p.  589. 

12  Jaderholm,  Zt.  f.  Biol.  Bd.  xiii.  (1877),  S.  193. 


227 


228  METH^MOGLOBIN. 

let  the  mixture  stand  for  a  short  time,  and  if  the  band  character- 
istic of  methsemoglobin  has  not  made  its  appearance,  add  one  or 
two  drops  more  of  the  solution  of  the  salt  and  proceed  as  before. 
As  soon  as  the  bands  of  oxy-hajmoglobin  have  markedly  disap- 
peared, acidulate  very  faintly  and  examine  again.  The  band 
which  should  now  be  visible  as  characteristic  of  methaemoglobin 
lies  in  the  red  part  of  the  spectrum,  between  C  and  D,  nearer  to 
the  former  line  As  already  remarked,  its  position  is  closely  sim- 
ilar to  that  of  hfematin  in  acid  solution  ;  but  comparison  will 
show  that  it  lies  nearer  D  than  does  the  hsematin  band,  the 
centre  of  the  latter  being  situated  at  w.  l.  640,  while  that  of  the 
former  is  at  w.  L.  630  ^  (See  Fig.  37,  Nos.  4  and  5). 

In  addition  to  the  reagents  recommended  above,  an  extensive  series 
of  other  substances  are  also  found  to  effect  the  conversion  of  oxy- 
liEemoglobin  into  methaemoglobin,  such  as  potasssium  chlorate,  amyl- 
nitrate,  iodine  dissolved  in  potassium  iodide,  bromine,  osmic  acid, 
hydrochinon,  pyrocatecliin,  &c.'^  It  may  also  be  obtained  as  the 
result  of  prolonged  evacuation  with  a  mercurial  pump,  of  putrefactive 
changes,  or  of  the  action  of  palladium  saturated  with  hydrogen  and 
immersed  in  the  solution  of  oxy-hgemoglobin.^ 

The  absorption  band  which  has  so  far  been  described  is  the  one 
which  is  to  be  regarded  as  characteristic  of  methtemoglobin,  being 
accompanied  by  a  very  marked  absorption  of  the  violet  end  of  the 
spectrum  extending  up  to  the  D  line.  In  addition  to  this  band  it 
is  stated  that,  working  with  a  good  spectroscope  of  low  dispersive 
power,  three  other  bands  may  be  additionally  seen,'*  two  corre- 
sponding closely  with  those  of  oxy-hsemoglobin  but  not  identical, 
their  centres  corresponding  to  w.  l.  580  and  539,  and  the  third  in 
the  blue  at  w.  L.  500  (?).5 

In  an  alkaline  solution  the  position  of  two  of  these  bands 
differs  slightly  from  that  just  given,  being  stated  by  Jiiderholm 
to  be  at  w.  l.  602  and  578,  while  the  third  is  unaltered  at 
539. 

In  the  preparation  of  large  quantities  of  crystallised  oxy- 
hsemoglobin  from  pig's  blood,  it  was  observed  that  during  the 
recrystallising  essential  to  its  purification  a  copious  crop  of 
reddish-brown  crystalline  needles  was  obtained.  These  were 
found  on  examination  to  be  crystals  of  methsemoglobin.^     They 

1  This  method  of  localising  the  bauds  means  that  their  centres  occupy  positions 
in  the  spectrum  where  the  wave-length  of  light  is  respectively  640  and  625 
millionths  of  a  millimeter.  It  should  always  be  adopted  for  all  absorption  bands, 
since  it  is  independent  of  the  varjang  dispersion  and  arbitrary  scales  of  different 
spectroscopes.     For  details  see  Gamgee,  Phi/siot.  Chem.  Vol.  i.  p.  94. 

■^  For  list  of  substances  see  Havem,  Compt.  Rend.  T.  cii.  (1886),  p.  698. 

3  Hoppe-Seyler,  Zt.  f.  physiol.  Chem.  Bd.  ii.  (1878),  S.  149. 

*  Jaderholra,  Zt.  j.  Biol.  Bd.  xx.  (1884),  S.  419.  Also  Nord.  Med.  Arkiv. 
Abst.  in  Maly's  Jahresb.  1884,  S.  113.  But  see  also  Araki,  Zt.  f.  physiol.  Chem. 
Bd.  XIV.  (1890),  S.  405. 

^  For  figure  see  Halliburton,  Chem.  Physiol,  and  Pathol.  Fig.  59,  Spect.  6,  p.  277. 

s  Hiifuef  u.  Otto,  Zt. /.physiol.  Chem.  Bd.  vii.  (1883),  S.  65. 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.        229 

are  most  easily  obtained  if  the  oxy-hsemoglobin  is  first  converted 
into  methaemoglobin  by  the  action  of  potassium  ferricyanide  (one 
or  two  minute  crystals  of  the  salt  to  half  a  litre  of  warm  con- 
centrated solution  of  oxy-hsemoglobin) ;  the  mixture  is  then 
shaken  until  it  has  a  dark -brown  colour  and  is  cooled  to  0°  after 
the  addition  of  one  quarter  of  its  bulk  of  alcohol  also  cooled  to 
0°.  They  have  also  been  obtained  from  the  blood  of  the  dog,^ 
horse,^  and  other  animals,^  and  resemble  in  crystalline  form  the 
crystals  of  oxy-hsemoglobin  from  the  same  sources.  These  crystals 
are  doubly  refracting,  readily  soluble  in  water,  though  less  so  than 
oxy-hsemoglobin,  and  the  solution,  unlike  that  of  the  latter  sub- 
stance, yields  a  precipitate  with  basic  lead  acetate  in  presence  of 
ammonia  ;  they  are  identical  in  percentage  composition  with  those 
of  oxy-hsemoglobin.  The  behaviour  of  methsemoglobin  towards 
reducing  agents  is  interesting  and  also  important  as  affording  a 
means  of  discrimination  between  this  substance  and  hsematin. 
If  some  ammonium  sulphide  be  added  to  an  alkaline  solution  of 
methsemoglobin,  the  mixture  may  be  observed  to  yield  the  spectrum 
of  (reduced)  hsemoglobin  ;  and  on  now  shaking  up  with  oxygen 
(air)  it  shows  the  spectrum  of  oxy-hsemoglobin.  When  a  solution 
of  hsematin  is  similarly  treated  it  yields  the  spectrum  of  hsemo- 
chromogen  (reduced  hsematin)  in  alkaline  solution  (see  below). 
While  the  close  relationship  of  methsemoglobin  to  oxy-hsemoglobin 
is  thus  clearly  shown,  very  great  differences  of  opinion  have  ex- 
isted as  to  the  exact  nature  of  that  relationship.  Three  views 
have  been  put  forward.  1.  That  methsemoglobin  is  more  highly 
oxidised  than  oxy-hsemoglobin.  2.  That  it  is  less  highly  oxidised. 
3.  That  it  is  united  with  exactly  the  same  amount  of  oxygen  as 
is  oxy-hsemoglobin,  only  in  a  more  stable  combination.  The  first 
view  seems  to  have  been  based  on  the  ready  production  of 
methsemoglobin  by  oxidising  agents,  and  on  the  statement  that 
when  methsemoglobin  is  reduced  it  yields  first  oxy-hsemoglobin 
and  then  hsemoglobin.  The  second  view  rested  on  the  possibility 
of  obtaining  methsemoglobin  by  the  prolonged  action  of  a  vacuum 
or  the  shorter  action  of  palladium  saturated  with  hydrogen, 
and  on  the  statement  that  by  reducing  agents  it  passes  at  once 
to  hsemoglobin  without  the  intermediate  appearance  of  oxy- 
hsemoglobin.  The  third  view,  which  now  appears  to  be  generally 
accepted,  is  derived  from  observations  of  the  amount  of  oxygen 
which  can  be  pumped  out  from  a  mixture  of  methsemoglobin  and 
oxy-hsemoglobin  of  known  composition,^  and  from  the  amount  of 

1  Hiifiier,  Ibid.  Bd.  viii.  (18841,  S.  366.  Jaderholm,  Zt.f.  Biol.  Bd.  xx.  (1884), 
S.  419. 

2  Hammarsten,  quoted  by  Jaderholm,  he.  cit.  S.  422. 

3  Halliburton,  Quart.  Jl.  Mic.  Sci.  Vol.  xxviii.  (1588),  p.  201.  Gives  rapid 
method  for  microscopic  purposes.     See  his  Chem.  Phi/siol.  and  Pathol,  p.  280. 

*  The  literature  of  the  dispute  is  fully  quoted  and  abstracted  down  to  1883  by 
Otto,  Pfliiger's  Arch.  Bd.  xxxi.  Sn.  245 — 2.5.5.  The  remaining  literature  to  date 
(1892)  has  been  given  passim  in  the  above  account  of  this  substance. 


230  H^MOCYANIN. 

oxygen  whicli  is  displaced  from  a  given  weight  of  metlisemoglobin 
when  it  is  treated  with  nitric  oxide.^  We  may  probably  say, 
therefore,  that  under  certain  conditions,  without  our  being  able 
to  state  exactly  what  has  taken  place,  the  oxygen  loosely  united 
to  hffimoglobin  as  oxy-hsemoglobin  becomes  more  stably  combined, 
and  is  now  not  removable  by  either  a  vacuum,  or  carbon-monoxide, 
or  a  current  of  hydrogen,  and  further  that  the  resulting  substance 
(methaemoglobin)  has  the  same  composition  and  crystalline  forms 
as  oxy-hajmoglobin,  and  may  be  reconverted  into  the  latter  body 
by  suitable  means,  such  as  reduction  by  ammonium  sulphide  and 
subsequent  oxidation. 

7.  Haemocyanin.2  As  previously  stated  (p.  217)  the  blood- 
plasma  of  many  invertebrates  contains  hemoglobin  in  solution ; 
in  a  few  cases  this  is  united  to  special  corpuscles  in  the  blood. 
But  in  the  case  of  other  invertebrates  this  respiratory  pigment  is 
replaced  by  another  to  which,  since  it  turns  blue  on  exposure  to 
air  (oxygen),  the  name  hsemocyanin  has  been  given.  Hence  the 
arterial  blood  of  those  animals  in  which  it  occurs  is  blue,  while  the 
venous  is  colourless. 

Hsemocyanin  is  a  proteid  of  the  globulin  class ;  it  is  therefore 
partially  precipitated  by  a  current  of  carbon-dioxide,  by  satura- 
tion of  its  solutions  with  sodium  chloride,  and  completely  by  satu- 
ration with  magnesium  sulphate.^  Unlike  heemoglobin  it  has  not 
yet  been  crystallised  and  contains  copper,  presumably  as  a  con- 
stituent of  its  molecule,  in  place  of  the  iron  characteristic  of 
haemoglobin.  It  exhibits  no  absorption  bands  when  examined 
spectroscopically. 

Another  animal  pigment  is  known,  into  whose  composition  copper 
(5 — 8  p.  c.)  enters  ;  this  is  the  substance  called  turacin.*  It  gives 
the  characteristic  colour  to  the  plumage  of  certain  African  birds  known 
as  Touracos  or  Plantain-eaters,  whence  the  name  turacin.  It  differs 
entirel}^  from  ha^mocj'anin  in  its  general  properties,  and  is  only  men- 
tioned here  because  it  contains  copper,  as  does  the  former  pigment. 
It  is  slightly  soluble  in  water,  readily  soluble  in  dilute  alkalis,  the 
solutions  in  either  of  these  solvents  showing  two  absorption  bands  be- 
tween D  and  HJ  very  similar  to,  though  not  identical  with,  the  bands 
of  oxy-hsemoglobin  and  a  third  faint  broad  band  at  F.  It  is  not  how- 
ever a  respiratory  pigment. 

1  Hlifner  u.  Kiilz,  Zt.f.  pJu/slol  Chem.  Bd.  rii.  (1883),  S.  366. 

2  For  literature  see  Halliburtou,  Chem.  Phi/sioi.  and  Pathol.  1891,  p.  321. 
Details  of  previous  work  to  date  (1880)  are  given  by  Krukenberg,  Vergleich.-ph>jsioI . 
Studien,  III.  Abth.  (1881),  S.  66. 

3  Halliburtou,  Jl.  ofPh)/siol.  Vol.  vi.  (1884),  p.  319. 

*  Church,  Phil.  Trans.  Vol.  CLix.  (1870),  p.  627.  Cf.  Ber.  d.  d.  chem.  Gesell.  Bd.  ii. 
(1869),  S.  314;  in.  (1870),  S.  459.  See  later  Krukenberg,  VergL-physiol.  Stud. 
V.  Abth.  (1881),  S.  72;  2  Reihe,  i.  Abth.  (1881),  S.  151.  The  same  work  (2  Reihe, 
Abth.  II.  II.  III.  1882,  Sn.  1  u.  128)  contaius  elaborate  observations  on  other  pigments 
from  feathers. 


CHEMICAL  BASIS   OF   THE  ANIMAL   BODY.        231 

8.    HsBmochromogen.     CgiHssNiEeOg  (?). 

When  (reduced)  hsemoglobin  is  treated  with  acids,  or,  better 
still,  with  alkalis  in  the  entire  absence  of  oxygen,  it  is  decom- 
posed into  a  proteid  and  a  coloured  substance  to  which  the  name 
hsemochromogen  was  first  given  by  Hoppe-Seyler.i  When  alkalis 
are  used  in  its  preparation,  the  solution  obtained  is  of  a  brilliant 
purplish-red  colour,  and  is  characterised  by  two  marked  absorp- 
tion bands,  the  stronger  lying  halfway  between  D  and  E,  the 
other  and  fainter  between  E  and  h.  These  are  identical  with 
the  bands  of  Stokes'  reduced  hsematin  in  alkaline  solution  (see 
Fig.  37,  No.  3).  When  exposed  to  the  air  (oxygen)  the  solution 
rapidly  loses  its  brilliant  colour,  becomes  dichroic,  viz. :  red  in 
thick,  and  greenish  in  thin  layers  (cf.  sub  hsematin)  and  now 
yields  an  absorption  spectrum,  which  exhibits  one  not  very 
strongly  marked  band  in  the  yellow,  to  the  red  side  of  D  and 
touching  the  latter  line.  This  is  the  spectrum  of  ha?matin  in  an 
alkaline  solution  (see  Fig.  37,  Nos.  1  and  2).  When  the  decom- 
position of  the  hsemoglobin  is  brought  about  by  acids  instead  of 
alkalis,  the  coloured  product  is  similarly  hsemochromogen,  but  in 
this  case,,  unless  special  precautions  are  taken,  some  of  the  hsemo- 
chromogen is  itself  further  decomposed  and  yields  hsematopor- 
phyrin  or  iron-free  hsematin  (see  below).  The  mixture  thus 
obtained  probably  accounts  for  the  four-banded  spectrum  as  first 
described  by  Hoppe-Seyler.''^  When  a  solution  of  hajmatin  in 
alkali  is  reduced  with  Stokes'  fluid  (see  sub  oxy -haemoglobin)  or 
ammonium  sulphide  the  solution  obtained  shows  two  absorption 
bands  identical  with  those  already  described  as  characteristic  of 
hasmochromogen.  From  these  facts  it  would  at  first  sight  appear 
that  reduced  hasmatin  in  alkaline  solution  and  ha^mochromogen 
in  a  similar  solution  are  identical  substances,  and  this  is  indeed 
the  view  which  has  been  most  generally  adopted.  From  a 
spectroscopic  point  of  view  they  do  appear  to  be  the  same,  but 
Hoppe-Seyler  maintains  that  they  are  not.^  According  to  him 
hsemochromogen  is  a  simple  product  of  the  decomposition  of 
haemoglobin,  while  hsematin  is  an  oxidised  product  which  differs 
from  true  oxy-hsemochroraogen  by  being  united  to  a  smaller 
amount  of  oxygen  than  is  the  former.  He  has  further  succeeded 
in  obtaining  not  only  hsemochromogen  in  a  crystalline  form,^  but 
also  a  compound  of  hsemochromogen  with  carbon-monoxide  ex- 
hibiting the  absorption  bands  of  carbon-monoxide  haemoglobin 
and  containing  the  same  amount  of  carbon-monoxide  united  to 

1  Med.-chem.  Unters.  Hft.  iv.  (1871),  S.  540.  Quoted  iu  detail  by  Gamgee. 
Physiol.  Chem.  Vol.  i.  p.  118.  See  also  later  Hoppe-Seyler,  Zt.  f.  physiol.  Chem. 
Bd'.  I.   (1877),  S.  138. 

2  Loc.  cit.  Cf.  Jaderholm  (Swedish),  Abst.  in  Maly's  Jahresb.  1874,  S.  104, 
1876,  S.  86.     But  see  Hoppe-Seyler,  Phijsiol.  Chem.  (1881),  S.  394. 

3  Zt.  f.  physiol.  Chem.  Bd.  xiii.  (1889),'  S.  477. 

4  By 'the  action  of  strong  caustic  soda  at  100°  in  the  entire  absence  of  oxygen. 


232'  H^MATIN. 

each  atom  of  iron  as  does  that  body,  whereas  hsematm  in  alkaline 
solution  will  not  unite  with  carbon-monoxide.  He  therefore 
considers  that  haemoglobin  is  a  compound  of  a  proteid  with  this 
hsemochromogen,  to  which  it  owes  its  colour,  and  that  it  is  with 
the  hsemochromogen  group  rather  than  with  haemoglobin  as  a 
whole  that  the  gases  are  united  in  the  formation  of  such  com- 
pounds as  oxy-haemoglobin  and  carbon-monoxide  haemoglobin. 
Further  investigation,  more  particularly  of  the  crystalline  haemo- 
chromogen,  is  needed  for  the  final  establishment  of  these  views. 

9.     Hsematin.     CsiHasK^FeOs.i 

When  oxy-haemoglobin  is  decomposed  by  either  acids  or  alkalis 
it  yields  a  proteid  and  a  coloured  substance  known  as  haematin. 
This  decomposition  may  take  place  in  old  blood-clots  or  extrav- 
asations and  is  readily  produced  by  the  action  of  either  gastric  or 
pancreatic  juice  on  oxy-haemoglobin,  so  that  haematin  is  frequently 
found  in  the  contents  of  the  alimentary  canal  and  in  the  faeces, 
more  especially  with  a  flesh  diet.  It  has  also  been  found  in  urine 
as  the  result  of  poisoning  with  sulphuric  acid  or  arseniuretted 
hydrogen. 

Freparation.  The  following  method  slightly  modified  after  Kiihne  ^ 
may  be  advantageously  employed,  and  yields  not  only  solutions  which 
show  strikingly  the  spectroscopic  appearances  of  haematin  in  acid  and 
alkaline  solution,  but  also  finally  a  fairly  pure  and  typical  sjoecimen 
of  lisematin  itself.  Defibrinated  blood  is  made  into  a  thin  paste  by 
mixture  with  potassium  carbonate,  and  is  then  evaporated  to  dryness 
on  a  water-bath.  The  dry  residue  is  powdered,  placed  in  a  flask,  and 
extracted  with  about  four  times  its  bulk  of  strong  alcohol  by  boiling 
on  a  water-bath.  The  deeply  coloured  extract  thus  obtained  is  poured 
off  and  the  residue  again  extracted  as  before  with  alcohol,  the  process 
being  repeated  as  long  as  any  colouring  matter  is  extracted.  The  ex- 
tracts are  mixed  and  filtered  and  form  a  strong  solution  (a)  of  haematin 
in  alkaline  alcohol.  A  portion  of  this  extract  may  be  kept  for  spectro- 
scopic examination.  The  remainder  is  strongly  acidulated  by  the  care- 
ful addition  of  sulphuric  acid,  any  precipitate  which  is  formed  is 
removed  by  filtration,  and  the  filtrate  (b)  provides  a  typical  solution 
of  hsematin  in  acid  alcohol.  A  portion  of  this  may  as  before  be  kept 
for  spectroscopic  examination.  The  remainder  is  made  alkaline  by 
the  addition  of  an  excess  of  ammonia  and  filtered;  the  filtrate  (c)  is, 
as  in  the  case  of  (a),  a  solution  of  hfematin  in  alkaline  alcohol,  but 
now  the  extraneous  salts  present  are  chiefly  those  of  ammonium.  The 
filtrate  (c)  is  finally  evaporated  to  dryness  on  a  water-bath,  extracted 
with  several  portions  of  boiling  water,  and  the  undissolved  residue  con- 
sists of  fairly  pure  hsematin.  This  should  finally  be  washed  with 
alcohol  and  ether  and  then  dried  for  a  prolonged  period  at  130-150° . 

To  obtain  pure  haematin  it  is  probably  better  to  prepare  it  from 
hsemin  whose  purity  as  a  mother  substance  can  be  ensured  at  the  out- 

1  Hoppe-Seyler,  Med.-chem.  Untersuch.  1871,  lift.  4,  S.  523. 

2  Physiol.  CAem.  1868,  S.  202. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        233 

set  by  the  fact  that,  unlike  hsematin,  it  is  readily  obtained  in  crystals. 
(See  below.)  The  hsemin  crystals  should  be  boiled  with  strong  acetic 
acid,  then  washed  with  water,  alcohol,  and  ether,  and  .dissolved  in 
dilute  caustic  potash.  The  solution  is  then  filtered,  precipitated  with 
hydrochloric  acid,  and  washed  with  boiling  water  until  the  washings 
are  shown,  as  tested  by  nitrate  of  silver,  to  be  free  from  hydrochloric 
acid.  The  residue  is  finally  dried  by  prolonged  heating  to  130  — 
150°.  1 

For  ordinary  purposes  hsematin  is  characterised  chiefly  by  the 
spectroscopic  appearances  of  its  solutions.  When  dissolved  in  an 
alkali  (ammonia,  as  in  solution  (c)  above)  it  shows  one  absorp- 
tion band  in  the  yellow  adjoining  D  to  the  red  side  of  this  line, 
while  at  the  same  time  there  is  great  absorption  at  the  blue  end 
of  the  spectrum  (Fig.  37,  Kos.  1  and  2).  On  treatment  with  a 
reducing  agent,  Stokes'  fluid  or  ammonium  sulphide,  this  band  is 
replaced  by  two  others  in  the  green,  of  which  the  one  nearest  D 
is  remarkably  dense,  the  other  less  sharply  defined.  Very  little 
absorption  of  the  red  end  is  observed  while  that  of  the  blue  is  as 
before  very  marked  (Fig.  37,  No.  3).  This  is  the  spectrum  of 
Stokes'  reduced  hsematin  and  is  identical  with  that  of  Hoppe- 
Seyler's  hsemochromogen.  The  two  substances  have  usually  been 
regarded  as  identical,  but  this  is  disputed  by  Hoppe-Seyler  (see 
above).  Alkaline  solutions  of  hsematin  are  strongly  dichroic, 
being  ruby-red  in  thick  layers  and  greenish  in  thin  layers 
viewed  by  reflected  light. 

The  acid  alcoholic  solution  of  hsematin  (solution  (&)  above)  is 
characterised  by  one  absorption  b^and  between  C  and  D,  adjoining 
C,  whose  centre  is  situated  at  w.  l.  640.  This  band  is  somewhat 
similar  to  that  of  methsemoglobin,  but  it  is  less  dense,  and  careful 
observation  shows  that  the  centres  of  the  respective  bands  do  not 
coincide  (Fig.  37,  Nos.  5  and  4).  Acid  solutions  of  hsematin  are 
monochromatic  and  of  a  dull  reddish-brown  colour.  If  blood  or  a 
strong  solution  of  oxy-hsemoglobin  be  made  strongly  acid  by  the 
addition  of  acetic  acid  the  hsemoglobin  is  decomposed,  hsematin  is 
set  free,  and  if  the  solution  be  shaken  up  with  ether  and  allowed 
to  stand,  the  ether  rises  to  the  surface  and  is  more  or  less  coloured 
owing  to  the  presence  of  hsematin  held  in  solution  in  the  acid  ether. 
This  acid  ethereal  solution  shows,  in  addition  to  the  one  band  al- 
ready described  as  characteristic  of  hsematin  in  an  acid  solution, 
three  other  bands  whose  positions  and  relative  intensities  are  suf- 
ficiently shown  in  Fig.  37,  Xo.  6. 

Hsematin  as  prepared  by  the  methods  described  above  is  usu- 
ally obtained  as  a  scaly  but  not  crystalline  mass  of  bluish-black 
colour  and  metallic  lustre,  strongly  resembling  iodine.  When 
finely  powdered  it  appears  dark  or  light-brown  according  to  the 

1  Hoppe-Sevler,  Physiol.-pathol.-chem.  Anal.  5  Aufl.  1883,  S.  239.  See  also  Caze- 
neuve,  TT^es^,  *Paris,  1876,  Abstr.  in  Maly's  Jahresh.  1876,  S.  76.  Bull.  Soc.  Chim. 
T.  XXVII.  (1877),  p.  485.     MacMunn,  Jl.  of  Physiol.  Vol.  vi.  1884,  p.  22. 


234  HISTOHiEMATmS. 

fineness  of  the  powder.  It  is  a  remarkably  stable  substance  ; 
may  be  heated  to  180°  without  decomposition,  but  by  stronger 
heating  is  finally  decomposed,  liberates  an  odour  of  hydrocyanic 
acid,  and  leaves  a  residue  (12-5  p.  c.)  of  pure  oxide  of  iron.  It  is 
quite  insoluble  in  either  water,  alcohol,  ether,  chloroform,  or  ben- 
zol. It  is  somewhat  soluble  in  strong  acetic  acid,  especially  if 
warm,  also  in  alcohol  (not  water)  to  which  some  acid  has  been 
added,  and  readily,  soluble  in  alkaline  solutions  or  in  alcohol  con- 
taining alkalis.  It  is  not  affected  either  by  strong  caustic  alkalis 
even  when  heated,  or  by  hydrochloric  or  nitric  acids.  It  may  be 
dissolved  in  strong  sulphuric  acid,  but  is  now  found  to  have  un- 
dergone a  change  during  solution  which  results  in  the  removal  of 
iron  and  the  production  of  hsematoporphyrin  or  iron-free  lisema- 
tin  ^  (see  below). 

If  the  decomposition  of  hsematin  by  sulphuric  acid  be  brought  about 
in  the  absence  of  oxygen  an  iron-free  insoluble  substance  is  obtained 
known  as  htematolin,  to  which  the  formula  CggHygNgOy  is  assigned.^ 

If  potassium  cyanide  be  added  to  an  alkaline  solution  of  heematin, 
this  now  shows  one  broad  absorption  band  extending  from  D  to  E 
(Hoppe-Seyler).  By  the  action  of  reducing  agents,  this  band  is  re- 
placed by  two  other  bands. ^  The  substance  to  which  these  appear- 
ances are  due  is  known  as  cyan-hsematin,  but  all  further  information 
is  still  wanting. 

Some  more  recent  observers  (Nencki  and  Sieber)  have  assigned 
to  hsematin  the  formula  C32ll32]Sr4Fe04,  the  validity  of  which  as 
against  the  views  of  Hoppe-Seyler  is  not  as  yet  generally  accepted. 
It  will  be  referred  to  again  under  hsemin. 

10.  Histohaematins.  This  is  the  name  assigned  to  a  class  of 
pigments  which  are  stated  to  be  of  wide-spread  occurrence  in  the 
tissues  of  both  vertebrates  and  invertebrates,  and  to  be  related  to 
though  quite  distinct  from  hsemoglobin  and  hsematin.  They  are 
regarded  as  respiratory  pigments,  playing  towards  the  muscles  or 
other  tissues  in  which  they  more  particularly  occur  the  same  part 
that  haemoglobin  does  to  the  tissues  generally.  Our  knowledge  of 
these  pigments  is  however  as  yet  limited  to  the  spectroscopic  ap- 
pearances which  they  present  either  in  situ  in  the  mother-tissue 
or  in  solutions  obtained  by  the  action  of  ether,  while  their  respi 
ratory  function  is  assumed  from  the  changes  which  they  exhibit 
under  the  influence  of  reducing  agents  and  subsequent  exposure 
to  oxygen.  Of  these  histohaematins  the  one  most  fully  de- 
scribed is  known  as  myohsematin  from  its  characteristic  presence 
in  muscles. 

1  The  haematoin  of  Preyer.     See  "  Die  Blutkrystalle,"  1871,  S.  178. 

2  Hoppe-Seyler,  Med.-chevi.  Unters.  1871,  Hf.  4,  S.  533.  Cf.  Nencki  u.  Sieber, 
Ber.  d.  d.  ckem.  Gesell.  Bd.  xvii.  (1884),  S.  2272. 

3  See  Gamgee,  Physiol.  Chem.  Vol.  i.  p.  115. 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.        235 

Myohcematin}  To  observe  the  spectrum  of  this  substance  a 
slice  of  tissue,  such  as  that  of  the  heart,  is  squeezed  in  a  com- 
pressorium  until  sufficiently  thin  to  transmit  light.  It  is  then 
examined  with  a  microspectroscope  under  strong  illumination. 
Or,  on  the  other  hand,  the  tissue  may  be  treated  with  excess  of 
ether  under  whose  influence  an  aqueous  juice  is  extruded  in 
which  the  myohsematin  is  in  solution.  Speaking  generally,  for 
the  appearances  vary  slightly  according  to  the  source  of  the  pig- 
ment, myohsematin  yields  a  four-banded  absorption  spectrum. 
The  first  band  lies  close  to  D,  but  towards  the  red  end  of  the 
spectrum.  The  next  two  bands  are  situated  close  together  about 
midway  between  D  and  E.  The  remaining  baud  lies  in  the  re- 
gion between  E  and  h.  Solutions  of  myohsematin  are  when  weak 
of  a  reddish-yellow  colour,  but  if  strong  they  are  pure  red.  By 
the  action  of  warm  alcohol  containing  a  little  sulphuric  acid  a 
spectrum  is  obtained  closely  similar  to  that  of  hajmatin  in  acid 
solution,  and  by  the  use  of  concentrated  sulphuric  acid  a  sub- 
stance is  produced  which  in  both  acid  and  alkaline  solutions  shows 
bands  similar  to  those  of  hsematoporphyrin  in  the  same  solvents. 
Under  certain  conditions  myohsematin  becomes  '  modified  '  and  now 
yields  two  bands  similar  to  those  of  hsemochromogen,  but  situated 
nearer  the  violet  end  of  the  spectrum. 

The  conclusions  drawn  from  the  above  spectroscopic  facts  have 
been  the  subject  of  some  controversy  and  adverse  criticism,  the 
appearances  being  regarded  as  due  not  to  a  specific  pigment,  but 
rather  to  hsemochromogen  or  mixtures  of  other  products  of  the 
decomposition  of  hsemoglobin.^ 

11.  Hsemin.  C34H35lsr4Fe05 .  HCl.  (Hsematin-hydrochloride, 
or  Teichmann's  crystals.) 

These  crystals  may  be  readily  obtained  for  microscopic  ex- 
amination by  heating  a  drop  of  fresh  blood  on  a  glass-slide  under 


Fig.  38.     H^min  crystals  from  a  drop  of  blood.     (Kiihne.) 

a  cover-slip  with  a  little  glacial  acetic  acid.^     In  the  case  of 
blood  which  has  been  dried,  as  in  an  old  blood-clot  or  stain,  the 

1  MacMunn,  Phil.   Trans.  Pt.  i.  1886,  p.  267,  JZ.  of  Physiol.  Vol.  viii.  (1887),' 
p.  .51. 

2  Levy,  Zt.  f.  -physiol.  Chem.  Bd.  xiii.  (1889),  S.  309.     Hoppe-Seyler,  Ibid.  Bd. 
XIV.  (1890),  S.  106.    'For  reply  see  MacMunn,  Ibid.  xiii.  S.  497,  xiv.  328. 

3  Teichmann,  Zt.  f.  rat.  Med.  Bd.  in.  (1853),  S.  375,  Bd.  viii.  S.  141. 


236  H^MIN. 

residue  should  be  powdered  as  finely  as  possible  with  a  minute 
quantity  (trace)  of  sodium  chloride.  A  little  of  the  powder  is 
then  placed  on  a  slide  and  covered  with  a  slip  under  which  some 
glacial  acetic  acid  is  now  run  in.  It  is  then  warmed  carefully  to 
a  temperature  just  short  of  that  which  would  cause  the  acid  to 
boil.  If  the  operation  has  been  successful,  on  cooling  crystals  of 
hsemin  will  be  seen  under  a  microscope,  mixed  in  either  case  as  in 
Fig.  38  with  a  granular  ddbris.  If  they  are  absent,  warm  again, 
adding  more  acid  if  necessary.  The  crystals  are  dark-brown,  fre- 
quently almost  black,  elongated  rhombic  plates  and  prisms  be- 
longing to  the  triclinic  system.^     In  a  purified  specimen  they  are 


Fig.  39.     HiEMiN  crystals.     (After  Preyer.) 

arranged  singly  or  in  groups  as  shown  in  Fig.  39,  and  apart  from 
their  form  are  characterised  by  being  strongly  doubly -refracting : 
when  examined  under  the  microscope  between  crossed  Mcol 
prisms  those  crystals  whose  axes  are  suitably  inclined  to  the  in- 
cident light  stand  out  bright  yellow  or  orange  on  the  dark  field.^ 
They  are  quite  insoluble  in  either  water,  alcohol,  ether,  chloro- 
form, or  dilute  acids :  they  may  however  be  dissolved  to  some 
extent  in  glacial  acetic  or  hydrochloric  acids,  especially  if 
warmed,  and  are  readily  soluble  in  alkaline  carbonates  or  dilute 
caustic  alkalis,  being  at  the  same  time  decomposed  by  the  latter 
solvent  into  hsematin  and  a  chloride  of  the  alkali.  This  fact  pro- 
vides the  best  means  for  obtaining  pure  hsematin  (see  above). 

Although  it  is  quite  easy  to  obtain  typical  crystals  under  the 
microscope  from  minute  amounts  of  haemoglobin  or  hsematin,  their 
preparation  on  a  large  scale  is  somewhat  tedious  ;  several  methods 

1  Lahorio.  Quoted  by  Schalfejew,  Jn.  d.  russ.  phys.-chem.  Gesell.  1885,  S.  30. 
See  Abstr.  in  Ber.  d.  d.  chem.  Gesell.  Bd.  xviii.  Ref.,  S.  232.  Cf.  Hogyes,  Centralh. 
f.  d.  med.  Wiss.  1880,  No.  16. 

2  A.  Ewald,  Zt.f.  Biol.  Bd.  xxir.  (1886),  S.  474. 


CHEMICAL  BASIS   OF  THE   AKIMAL  BODY.         237 

have  been  employed,^  of  which  the  most  recent,  said  to  yield  5  gr. 
of  crystals  from  each  1  litre  of  blood,  is  as  follows.^  To  each 
volume  of  defibrinated  and  strained  blood  add  four  volumes  of 
glacial  acetic  acid  previously  warmed  to  80°.  As  soon  as  the 
temperature  of  the  mixture  has  fallen  to  55 — 60°,  it  must  be 
again  warmed  to  80°.  On  cooling  and  standing  for  10  — 12  hours 
crystals  separate  out ;  the  supernatant  liquid  is  then  removed  by 
a  syphon,  the  crystals  are  washed  with  water  repeatedly  by  de- 
cantation  in  a  tall  glass  cylinder  and  are  finally  collected  on  a 
filter  and  washed  with  water,  alcohol,  and  ether. 

The  successful  preparation  of  hsemin  crystals  from  minute 
quantities  of  haemoglobin  or  methsemoglobin  is  of  the  greatest 
importance  for  medico-legal  purposes,  since  they  suffice,  even  in 
the  absence  of  all  other  confirmatory  evidence,  to  establish  the 
nature  of  the  material  used  in  their  preparation.  In  the  detection 
of  blood-stains  it  is  usual  first  to  examine  with  a  spectroscope  an 
aqueous  solution  of  the  colouring  matter  if  it  can  be  obtained,  for 
the  characteristic  absorption  bands  of  oxy-hsemoglobin  or  methsemo- 
globin.  In  old  stains  the  haemoglobin  is  frequently  decomposed, 
in  which  case  it  is  insoluble  in  water,  and  alkaline  extracts  must 
be  made  and  examined  for  the  spectra  characteristic  of  hsematin. 
The  residues  from  the  spectroscopic  examination  are  lastly  used 
to  prepare  hsemin  crystals,  in  final  confirmation  of  the  evidence 
previously  obtained.^ 

Allusion  has  already  been  made  (see  p.  234)  to  some  work  on  hgemin 
and  heematin  which  assigns  to  these  substances  a  composition  and 
relationship  very  different  from  those  usually  accepted,  and  further 
puts  the  relationship  of  the  colouring  matter  of  blood  to  the  bile- 
pigments  in  a  new  light.'*  With  the  preliminary  caution  that  these 
views  are  not  as  jet  generally  accepted  and  require  confirmation,  they 
may  be  briefly  dealt  with  here.  Using  amyl-alcohol  in  the  prepara- 
tion of  hsemin  crystals  it  is  stated  that  the  crystals  have  the  following 
composition  (C32H3oN4Fe03  .  HC1)4  C5H9 .  OH.  The  group  C32H30N4 
FeOa  is  regarded  as  the  true  hsemin,  Teichmann's  crystals  consisting 
of  C32H3oN4Fe03 .  HCl.  When  the  crystals  thus  prepared  are  decom- 
posed by  caustic  alkalis  as  in  the  ordinary  method  for  preparing  hsema- 
tin  from  them,  the  haemin  is  supposed  to  take  up  one  molecule  of 
water  and  yield  hsematin  C32H32N4Fe04.  By  treating  this  hsematin 
with  strong  sulphuric  acid,  it  loses  its  iron  and  uniting  with  oxygen 
yields  hsematoporphyrin  or  iron-free  h^ematin,   C32H32]Sr405,  which  is 

1  See  Gamgee,  Phi/siol.  Chem.  Vol.  i.  p.  116,  or  Hoppe-Seyler,  Physiol,  patkol.- 
chem.  Anal  Aufl.  5,  1883,  S.  241. 

■•^  Schalfejew,  Jn.  d.  russ.  phi/s.-chem.  Gesell.  1885.  See  Abstr.  in  Ber.  d.  d. 
chem.  Gesell.  xviii.  Bd.  (1885),  Ref.,  S.  232. 

3  For  details  see  Hoppe-Seyler,  loc.  cit.  S.  529.  Gamgee,  loc.  cit.  p.  217. 
MacMunn,    The  spectroscope  in  medecine,   1883,  pp.    130 — 148. 

*  Nencki  u.  Sieber,  Ber.  d.  d.  chem.  Gesell.  Bd.  xvii.  (1884),  S.  2267,  xviii. 
S.  392,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  xviii.  (1884),  S.  401,  Bd.  xx.  (1886),  S.  325. 
Bd.  XXIV.  (1888),  S.  430.  Nencki  u.  Rotschy.  Monatshf.  f.  Chem.  Bd.  x.  (1889), 
S.  568.  See  also  Hoppe-Sevler  in  adverse  criticism,  Ber.  d.  d.  chem.  Gesell.  Bd.  xviii. 
(1885),  S.  601,  Zt.f.  physiol.  Chem.  Bd.  x  (1886),  S.  331. 


238  H^MATOPOEPHYEIN. 

however  further  regarded  as  derived  by  dehydration  from  a  true  hsema- 
toporphyrin  whose  composition  is  CisHigNjOs.  The  latter  is  thus 
identical  in  composition  with  bilirubin,  whose  formula  is  undoubtedly 
Ci6Hi8N203.  This  is  regarded  as  affording  the  desired  chemical  proof 
of  the  genetic  relationship  of  the  bile-  and  blood-pigments,  the  deri- 
vation of  the  former  from  hsematin  being  represented  as  follows, 
C32H32N4Fe04  +  2H2O  -  Fe  =  2  (CieHisN^Oa) . 

12.   Hsematoporphyrin.^  C68H74]Sr80i2(?).  (Iron-free  hsematin.) 

If  hsematin  is  dissolved  in  concentrated  sulphuric  acid  it  yields 
a  solution  which,  after  filtration  through  asbestos,  is  of  a  brilliant 
purple-red  colour.  By  the  action  of  the  acid,  the  iron  is  removed 
from  the  hsematin  and  hsematoporphyrin  is  formed.^  If  this  solu- 
tion is  diluted  with  sulphuric  acid  it  shows  with  a  spectroscope 
two  absorption  bands  of  which  one  adjoins  D  to  the  red  side  of 
this  line,  while  the  other  is  very  strongly  marked  and  lies  midway 
between  D  and  E.  By  the  addition  of  water  to  the  solution  in 
sulphuric  acid  the  colouring  matter  is  largely  precipitated,  especi- 
ally if  some  alkali  be  carefully  added  to  neutralise  the  acid.  The 
precipitate  thus  obtained  is  readily  soluble  in  dilute  alkalis,  and 
this  solution  is  characterised  by  four  absorption  bands,  one  half- 
way between  C  and  D,  two  between  D  and  E,  and  one  conspicuous 
band  adjoining  h  and  extending  nearly  to  F?  Hsematin  also  yields 
hfematoporphyrin  by  the  action  of  strong  hydrochloric  acid  at  130° 
in  sealed  tubes. 

Some  interest  attaches  to  this  substance  owing  to  its  occasional 
occurrence  in  urine  in  forms  which  show  slightly  different  absorp- 
tion spectra  but  are  probably  closely  related  if  not  identical. 
Thus  it  occurs  as  urohtematin  or  urohsematoporphyrin,*  or  as 
ordinary  hsematoporphyrin.^  It  is  also  found  in  the  integument 
of  some  invertebrates  ^  and  in  the  egg-shells  of  certain  birds.''  It 
is  further  interesting  to  notice  that  in  hsematoporphyrin  we  have  a 
strongly  coloured  pigment  derived  from  hsematin  with  removal  of 
the  iron  which  the  latter  contains,  a  fact  which  facilitates  our  con- 
ception of  a  possible  derivation  of  the  iron-free  bile-pigments 
from  the  iron-containing  hsemoglobin  or  hsematin.  This  relation- 
ship will  be  more  fully  discussed  when  the  bile-pigments  are 
described. 

1  Hoppe-Seyler,  Med.-chem.  Unters.  Hft.  4.  1871,  S.  528. 

2  In  the  absence  of  oxygen  a  substance  called  by  Hoppe-Sej'ler  hsematolin  is 
obtained,  CgsH^gFgOy. 

**  These  spectra  are  figured  in  Halliburton,  Chem.  Physiol,  and  Pathol.  Tig.  59, 
p.  277,  Nos.  10  and  11. 

*  MacMunn,  Proc.  Roy.  Soc.  Vol.  xxxi.  1880,  p.  206,  Jl.  of  Physiol.  Vols.  vi. 
(1884),  p.  36,  X.  (1889),  p.  71,  Clinical  Chem.  of  Urine,  1889,  p.  109,'  Proc.  Physiol. 
Soc.  No.  IV.  1890.     See  Jl.  of  Physiol.  Vol.  xi.  (1890),  p.  xiii. 

5  E.  Salkowski,  Zt.  f  physiol.  Chem.  Bd.  xv.  (1891),  S.  286. 

s  MacMunn,  Jl.  of  Physiol.  Vols.  vn.  (1885),  p,  240,  viii.  p.  384. 

■^  For  literature  see  MacMunn,  Jl.  of  Physiol.  Vol.  vii.  p.  251. 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.        239 

13.     Hcematoidin.^    CisHisNgOa. 

This  substance  is  found  as  reddish  or  orange  rhombohedral 
crystals  in  old  blood-clots  as  of  cerebral  haemorrhages,^  in  corpora 
lutea,  in  the  urine  in  cases  of  transfusion  of  blood  ^  and  in  cases 
of  hgematuria.*  There  is  no  doubt  that  as  occurring  in  the  above 
cases  it  is  directly  derived  from  some  metamorphosis  of  htemo- 
globin.  Apart  from  the  similarity  of  crystalline  form  and  colour 
it  was  further  found  that  hsematoidin  crystals  readily  give  the 
characteristic  (Gmelin's)  reaction  for  bilirubin  by  treatment  with 
nitric  acid,  and  thus  its  identity  with  bilirubin  was  at  once  asserted 
and  supported  by  very  convincing  evidence.^  The  identity  was 
however  for  some  time  disputed,  notably  by  Stadeler,   and  by 


Fig.  40.     H^MATOiDiN  Crystals.     (Frey  after  Funke.) 

others  largely  on  the  basis  of  inconclusive  spectroscopic  investi- 
gation of  the  two  substances.  There  is  however  no  doubt  that 
hsematoidin  is  really  identical  with  bilirubin,  so  that  now  the 
name  is  of  interest  rather  from  a  historical  point  of  view  and 
physiologically  as  indicating  the  undoubted  genetic  relationship 
of  the  pigments  of  bile  to  those  of  blood. 


BILE-PIGMENTS   AND   THEIE   DERIVATIVES.^ 

The  bile  is  in  all  animals  a  characteristically  highly-coloured 
secretion.     The  colour  of  the  fresh  bile  is  as  a  general  rule  golden- 

1  The  literature  of  this  substance  is  very  fully  quoted  in  Hermann's  Hdbch.  d. 
Physiol.  Bd.  v.  Th.  1.  S.  245.  . 

^  Virchow  first  carefully  described  it  as  obtained  from  this  source,  and  named  it 
heematoidin  to  indicate  its  undoubted  derivation  from  the  colouring  matter  of  the 
blood.     Virchow's  Arch.  Bd.  i.  (1847),  S.  419. 

3  Hoppe-Sevler,  Pfliiger's  Arch.  Bd.  x.  (187.5),  S.  211. 

4  Ebstein,  Deutsch.  Arch.f.  Klin.  Med.  1878,  S.  115. 

5  See  among  others  E.  "Salkowski,  Hoppe-Seyler's  Med.-chem.  Unters.  Hf.  3, 
1868,   S.   436. 


Also 
S.  497. 


See  specially  Maly  in  Hermann's  Hdbch.  d.  Physiol.  Bd.  v.  Th.  2,  1881,  S.  154. 
,  for  history  and  literature,  Heynsius  u.  Campbell,  Pfliiger's  Arch.  Bd.  iv.  (J 871), 


240  BILIEUBIN. 

red  in  man  and  carnivora,  and  more  or  less  bright  green  in  herbi- 
vora.  These  colours  are  due  to  the  presence  of  a  pigment  known 
as  bilirubin  in  the  first  case  and  biliverdin  in  the  second;  but 
since  the  latter  pigment  may  be  readily  formed  by  simple  oxida- 
tion from  the  former,  bile  may  frequently  contain  both  these 
colouring-matters  and  hence  possess  a  colour  intermediate  to  the 
above  though  usually  with  a  preponderance  of  either  the  golden- 
red  or  green  shade.  In  addition  to  these  two  pigments  others 
are  occasionally  present  in  bile,  as  evidenced  by  the  fact  that 
while  neither  bilirubin  nor  biliverdin  exhibits  any  absorption 
bands  when  examined  spectroscopically,  fresh  bile  of  herbivora  ^ 
frequently  does  show  bands,  due  to  substances  of  which  but  little 
is  known"  beyond  these  spectroscopic  appearances  (see  below).  It 
is  possible  that  the  bile-pigments  of  different  animals  may  ulti- 
mately be  found  to  differ  slightly  but  distinctly  in  their  composi- 
tion, much  in  the  same  way  that  the  bile-acids  as  already  stated 
differ ;  but  as  yet  no  such  distinct  differences  have  been  made  out, 
and  we  may  therefore  treat  of  them  as  being  identical  from  what- 
ever source  they  have  been  obtained. 

1.     Bilirubin.    CieHigNaOg.^ 

It  occurs  chiefly  and  characteristically  in  the  fresh  bile  of  man 
and  carnivora,  to  which  it  imparts  the  well-known  golden-red 
colour.  It  frequently  constitutes  the  larger  part  of  some  kinds 
of  gall-stones,  more  especially  of  the  ox  and  pig,  not  as  free  bili- 
rubin but  as  a  compound  with  chalk,  and  amounting  to  some  40 
p.c.  of  the  concretions.  (Maly.)^  It  is  also  found  in  the  urine 
in  icterus,  also  constantly  in  the  serum  from  horses'  blood,  though 
not  from  that  of  man  or  the  ox,'*  and  frequently  as  crystals  under 
the  name  '  htematoidin '  (see  above)  in  old  blood-clots  (extrava- 
sations) and  fluids  from  ovarial  and  other  cysts.  Bile-pigments 
are  also  stated  to  occur  normally  in  the  urine  of  dogs,  more  par- 
ticularly in  the  summer.^ 

Bilirubin  is  insoluble  in  water  and  almost  insoluble  in  either 
ether  or  alcohol,  though  distinctly  more  soluble  in  alcohol  than 
in  ether.  It  is  on  the  other  hand  readily  soluble  in  alkaline  solu- 
tions, hence  its  solution  in  bile,  also  in  glycerin  carbon-disulphide, 

1  Bile  of  carnivora  does  not  usually  show  bands  until  it  has  been  treated  with  au 
acid. 

2  This  is  the  generally  accepted  formula,  assigned  to  this  substance  by  Maly.  Jn. 
f.  prakt.  Chem.  Bd.  civ.  (1868),  S.  28,  confirming  Staedeler.     It  is  possible  that  the 

formula  is  really  twice  the  above,  viz.  C32H3,N40e,  as  required  to  represent  the 
formula  of  a  well-defined  tribromo-substitution  product,  C32H33Br3N406_.  _  This 
doubling  of  the  formula  is  also  necessary  to  express  the  derivation  of  hydrobilirubiii. 
(C32H40N4O7)  from  bilirubin.  Maly,  Sitzb.  d.  k.  Akad.  d.  Wiss.  Wien.  in.  Abth. 
Oct"-Hft.  1875.     Liebig's^Kwa/.  Bd.  CLXXXi.  (1876),  S.  106. 

3  See  earlier  Staedeler,  Vierteljahrschr.  d.  naturforsch.  Gesell.  Ziirich,  Bd.  viii. 
1863,  and  Liebig's  Annai.  Bd.  cxxxii.  (1864),  S.  323. 

4  Hammarsten  (Swedish).     See  Abstr.  in  Maly's  Jahresb.  1878,  S.  129. 

5  Salkowski  u.  Leube,  Die  Lehre  vom  Ham,  1882,  S.  246. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.        241 

and  benzol,  and  above  all  in  chloroform.  Troni  its  solution  in 
the  latter  it  may  be  separated  out  by  extremely  slow  evaporation 
of  the  solvent  in  a  crystalline  form  as  rhombic  plates  or  prisms. 
The  general  shape  of  these  is  shown  above  in  Fig.  40  ;  but  as 
obtained  from  solution  in  either  carbon-disulphide  or  chloroform 
the  crystals  usually  exhibit  somewhat  blunt  ends  and  slightly 
convex  surfaces  as  first  pointed  out  by  Staedeler.     As  ordinarily 


Fig.  41.    Bilirubin  Crystallised  from  Carbon-disulphide.   (Krukenberg.) 

prepared  it  is  an  amorphous  powder  of  the  colour  of  sulphide  of 
antimony.  It  readily  forms  compounds  with  bases,  e.  g.  sodium, 
barium,  and  calcium,  the  latter  providing  a  convenient  means 
for  the  separation  of  bilirubin  from  bile,  urine,  or  other  dilute 
solution. 

Preparation,  (i)  When  gall-stones  are  not  available  bile  may 
be  treated  as  follows.  ^  The  bile  is  slightly  diluted  with  water, 
some  lime-water  is  added  (avoiding  excess)  and  after  thorough 
mixture,  as  by  shaking,  a  current  of  carbon  dioxide  is  passed  to 
convert  all  the  excess  lime  into  carbonate.  The  precipitate  thus 
formed  contains  the  bilirubin  as  a  calcium  compound.  This  is 
then  collected  on  a  filter,  washed  with  water,  and  after  suspension 
in  a  little  water,  decomposed  by  the  addition  of  a  slight  excess  of 
acetic  or  hydrochloric  acid.  By  this  means  the  bilirubin  is  set 
free,  and  may  now  be  extracted  by  shaking  with  an  excess  of 
chloroform.  The  chloroform  solution  is  separated  by  decantation, 
and  evaporated  to  a  small  bulk ;  the  bilirubin  may  then  finally 
be  precipitated  by  an  excess  of  alcohol.  The  amount  thus  ob- 
tained is  not  quantitatively  accurate,  since  all  the  bilirubin  is  not 
precipitated  by  the  lime  at  the  outset  and  there  is  a  further  loss 
during  the  subsequent  operations,  (ii)  Since,  as  already  stated, 
the  gall-stones  of  the  ox  or  pig  may  consist  of  nearly  half  their 
weight  of  bilirubin  combined  with  calcium,  they  provide  the  best 

'  Based  on  Huppert,  Arch.  d.  Heilk.  Bd.  viii.  (1867),  S.  345,  476.  See  Hoppe- 
Seyler,  Hdbch.  d.  physiol.-path.  chein.  Anal.  1883,  S.  250.  Cf.  Hilger,  Arch.  d. 
Pharm.  (3),  Bd.  vi.  (1875),  S.  385. 

16 


242  BILIKUBIK 

and  simplest  source  for  the  preparation  of  this  substance.^  The 
stones  are  finely  powdered,  extracted  with  ether  to  remove  any 
cholesterin,  then  with  water  and  treated  with  either  strong  acetic 
acid  or  dilute  hydrochloric  acid.  By  this  means  the  bilirubin  is 
set  free  from  its  calcium  compound,  and  after  being  washed  with 
water  and  alcohol  is  dissolved  in  chloroform,  and  finally  separated 
by  precipitation  with  alcohol  as  already  described.  To  obtain  it 
quite  pure  the  dissolving  in  chloroform  and  precipitating  by  alco- 
hol should  be  repeated  several  times.  The  final  product  is  amor- 
phous. Crystals  are  most  readily  obtained  by  slow  evaporation 
of  the  first  and  hence  slightly  impure  solution  in  chloroform. 

When  carnivorous  bile  is  exposed  to  the  air  it  turns  more  or 
less  rapidly  green ;  this  is  due  to  its  oxidational  conversion  into 
biliverdin,  the  normal  pigment  of  herbivorous  bile.  A  similar 
change  is  at  once  produced  by  an  oxidising  agent  such  as  nitric 
acid  containing  nitrous  acid,  but  in  this  case  the  change  of  colour 
does  not  stop  short  with  green,  but  passes  successively  through 
blue,  violet,  and  red  to  a  final  yellow.  These  later  colours  are 
due  to  products  of  the  progressive  oxidation  of  the  first  formed 
biliverdin,  but  with  the  exception  of  the  final  substance  (cholete- 
lin)  are  as  yet  but  imperfectly  characterised.  The  play  of  colours 
observed  when  either  bilirubin  or  biliverdin  is  oxidised,  consti- 
tutes the  well-known  Clmelin's  reaction.^  This  is  extremely 
delicate  and  may  be  applied  in  either  of  the  two  following  ways. 
A  few  drops  of  the  suspected  solution  are  placed  on  a  porcelain 
slab  and  a  drop  of  yellow  fuming  nitric  acid  is  brought  into  con- 
tact with  it.  A  play  of  colours  is  observed  at  the  junction  of  the 
fluids  if  bile-pigments  are  present.  Or  on  the  other  hand  some 
of  the  acid  may  be  poured  into  the  bottom  of  a  test-tube  and  the 
suspected  fluid  carefully  added  so  as  not  to  mix  with  the  acid  but 
float  on  its  surface.  If  bile-pigments  are  present  coloured  rings 
(layers)  appear  at  the  junction  of  the  two  liquids,  being  yellow 
nearest  the  acid  and  progressively  red,  violet,  blue,  and  green 
passing  upwards.  It  is  stated  that  this  test  will  detect  as  little 
as  1  part  of  bilirubin  in  70,000  —  80,000  parts  of  solvent. 

Other  tests  have  been  recommended,  but  they  are  perhaps  un- 
necessary in  view  of  the  extreme  delicacy  of  Gmelin's  reaction  when 
properly  applied.^  The  certain  detection  of  minute  amounts  of  bile- 
pigments  in  urine  is  frequently  of  great  clinical  and  physiological 
importance.  If  any  very  appreciable  quantity  of  the  pigments  are 
present,   Gmelin's  reaction  applied  as   above  will  usually  suffice  to 

1  The  coloured  residue  from  human  gall-stones  left  after  the  extraction  of 
cholesterin  (p.  131)  may  also  be  used  for  the  preparation  of  bilirubin. 

-  Tiedemann  u.  Gmelin.     Die  Verdauung  nach  Versuchen,  1826,  S.  80. 

3  See  more  particularly  Capranica,  Gaz.  chim.  ltd.  Vol.  xi.  (1881),  p.  430. 
Moleschott's  Untersuch.  z.  Natmiehre,  Bd,  xiii.  (1882),  S.  190.  Ehrlich.  Centralb. 
f.  klin.  Med.  1884,  No.  45,  or  Centralb.  /.  d.  med.  Wisa.  1884,  S.  143.  In_  the  latter 
case  a  solution  of  diazobenzosulphonic  acid  is  employed,  and  is  stated  to  discriminate 
between  bilirubin  and  other  bile-pigments. 


CHEMICAL   BASIS   OF   THE  ANIMAL  BODY.        243 

detect  them.  If  not  they  may  be  obtained  in  a  more  concentrated 
residue,  which  has  been  largely  freed  by  Huppert's  method  from 
other  colouring  matters  which  interfere  with  the  test.  The  fluid  is 
precipitated  by  lime-water  and  carbon  dioxide.  The  compound  of 
lime  and  bilirubin  is  then  collected  on  a  filter,  washed  and  tested  in 
situ  by  the  addition  of  fuming  nitric  acid;  or  it  may  be  boiled  in  a 
test-tube  with  a  little  alcohol  acidulated  with  sulphuric  acid;  the  pre- 
cipitate loses  its  colour  and  the  supernatant  alcohol  turns  to  a  brilliant 
green.  The  following  is  also  a  reliable  test  as  applied  to  urine. -^  To 
20  or. 30  c.c.  of  urine  add  5  to  10  c.c.  of  a  solution  of  zinc  acetate 
(1:5).  This  causes  a' voluminous  precipitate  of  bile-pigments,  espe- 
cially if  the  acid  reaction  be  somewhat  reduced  by  the  simultaneous 
addition  ,of  a  little  sodium  carbonate.  The  precipitate  is  collected 
on  a  filter,  washed  with  water,  and  dissolved  in  a  little  ammonia.  If 
bile-pigments  are  present  the  solution  is  usually  fluorescent,  and  on 
standing,  if  not  at  once,  shows  the  absorption  bands  characteristic 
of  bilicj^anin.  (See  below.)  For  further  details  of  other  methods 
consult  some  special  work.^ 

The  accurate  quantitative  determination  of  bilirubin,  as  of 
other  bile,  and  also  of  urinary-pigments  is  only  possible  by 
spectrophotometric  methods.  These  have  been  already  briefly 
described  on  p.  224.  The  requisite  constants  for  the  application 
of  the  method  in  the  case  of  each  pigment  are  given  in  the  litera- 
ture quoted  below. ^ 

Bilirubin,  while  it  exhibits  no  distinct  absorption  bands,  is 
characterised  by  a  powerful  absorption  of  the  violet  end  of  the 
spectrum. 

2.     Biliverdin.     CisHigNaOi. 

This  is,  as  already  stated,  the  first  product  of  the  oxidation  of 
bilirubin.  It  gives  the  characteristic  colour  to  the  bile  of  herbi- 
vora,  probably  accounts  for  the  colour  of  biliary  vomit  in  carni- 
vora  (man),  is  possibly  found  in  the  urine  in  icterus,  has  been 
stated  to  occur  in  the  edges  of  the  placenta  in  pregnant  animals  * 
(bitches),  while  on  the  other  hand  it  occurs  in  mere  traces  in  gall- 
stones whether  of  man  or  other  animals.  It  has  also  been 
described  as  occurring  in  egg-shells^  and  the  integuments  of 
certain  invertebrates.^ 

Preparation.  An  impure  product  may  be  obtained  as  follows 
from  herbivorous  bile.     After  the  removal  of  mucin  (p.  76),  barium 

1  Stokvis.     See  Abst.  in  Maly's  Jahresb.  1882,  S.  226. 

-  Neubauer  u.  Vogel,  Anal.  d.  Hams,  1890,  S.  321  et  seq. 

3  Vierordt,  Die  quant.  Spectralanalyse  u.  s.  w.  Tubingen,  1876,  S.  76.  Zt.  f. 
Biol.  Bd.  IX.  (1873),  S.  160,  Bd.  x.  (1874),  S.  21,  399.  Vossius,  Arch.  f.  exp.  Pathol. 
Bd.  XI.  (1879),  S.  427. 

1  Etti.     See  Maly's  Jahresb.  1871,  S.  233,  and  1872,  S.  287. 

5  Liebermann,  Ber.  d.  d.  chem.  Ge.sell.  Bd.  xi.  (1878),  S.  601.  Krukeuberg, 
Verhandl.  d.  physik.-med.  Gesell.  zu  WUrzburg,  Bd.  xvii.  (1883),  S.  109. 

6  Krukenberg,  Cenlralb.f.  d.  med.  Wiss.  1883,  S.  785. 


244  BILIVEEDIK 

chloride  is  added ;  this  precipitates  the  pigment  as  a  compound 
with  barium  (?).  The  precipitate  is  then  collected  on  a  filter, 
washed  with  water  and  alcohol,  and  decomposed  with  dilute 
hydrochloric  acid ;  this  liberates  the  biliverdin  which  is  simultan- 
eously precipitated  as  a  flocculent  mass,  and  is  then  washed  with 
ether  to  remove  all  fat  and  dissolved  in  alcohol.  The  alcoholic 
solution  is  finally  filtered  and  by  spontaneous  evaporation  yields 
a  dark-green  glittering  residue  of  impure  biliverdin.  To  obtain 
the  pigment  pure  it  must  be  prepared  from  bilirubin.  The  con- 
version may  be  effected  in  several  ways.^  (i)  Bilirubin  is  dissolved 
in  a  dilute  alkali  and  exposed  for  some  time  to  the  air  in  thin 
layers,  whereby  it  is  slowly  oxidised  into  biliverdin.  When  the 
conversion  is  complete,  the  pigment  is  precipitated  by  the  addition 
of  hydrochloric  acid,  thoroughly  washed  with  water,  dissolved  in 
absolute  alcohol,  and  precipitated  by  an  excess  of  water  or  by 
spontaneous  evaporation  of  the  alcoholic  solution,  (ii)  By  en- 
closing bilirubin  solutions  in  tubes  with  glacial  acetic  acid  and 
heating  to  100''.  (iii)  By  the  action  of  monochloracetic  acid  and 
gentle  heating  at  intervals  for  one  or  two  days,  (iv)  Also  by  the 
action  of  caustic  potash  on  tribromobilirubin.^ 

Apart  from  its  colour  biliverdin  differs  most  characteristically 
from  bilirubin  in  its  solubilities.  It  is  (like  bilirubin)  soluble  in 
alkalis  but  insoluble  in  water  and  ether,  whereas  (unlike  bilirubin) 
it  is  insoluble  in  either  chloroform,  carbon  bisulphide  or  benzol, 
but  readily  soluble  in  alcohol  and  in  glacial  acetic  acid.  It  has 
further  never  been  obtained  in  a  crystalline  form,  and  like  bili- 
rubin it  shows  no  absorption  bands  but  a  somewhat  strong  absorp- 
tion of  the  violet  end  of  the  spectrum.  Treated  with  fuming 
(yellow)  nitric  acid  it  gives  Gmelin's  reaction,  starting  now  with 
a  blue  colour  as  a  product  of  the  first  stage  of  its  oxidation.  It 
also  yields  Huppert's  reaction.     (See  above  sub  bilirubin.) 

Like  bilirubin  the  quantitative  determination  of  biliverdin  is 
dependent  upon  spectrophotometric  methods.^ 

The  formula  assigned  above  to  biliverdin  represents  its  forma- 
tion from  bilirubin  by  simple  oxidation.*  This  is  undoubtedly 
correct  as  against  the  older  view  of  Staedeler  that  the  change 
consists  not  only  in  the  assumption  of  oxygen  but  also  of  a  mole- 
cule of  water. 


Bilifusoin,  bilihumin,  and  biliprasin  are  the  names  given  by  Staede- 
ler to  ill-defined  and  probably  impure  products  obtained  during  his 
investigations  on  bile-pigments  as  obtained  from  gall-stones.  Bili- 
prasin is  apparently  only  impure  biliverdin  (Maly). 


1  Maly,  Sitzh.  d.  k.  Akad.  Wien,  Bd.  lxx.  3  Abth.  1874.     Juli-Hft. 

2  Maly,  Ibid.  Bd.  lxxii.  3  Abth.  1875.     Oct.-Hft. 

3  See  references  sub  bilirubin. 

*  Maly,  loc.  cit.     Thudichum,  Jl.  Chem.  Soc.  July,  1876. 


CHEMICAL  BASIS   OF   THE  ANIMAL   BODY.        245 

3.  Bilicyanin.^     (Cholecyanin,  Choleverdin.) 

This  is  the  substance  which  results  from  the  oxidation  of  bili- 
verdin  and  is  the  cause  of  the  blue  colour  observed  when  bile  is 
treated  with  fuming  (yellow)  nitric  acid  as  in  Gmelin's  reaction. 
It  has  not  as  yet  been  isolated  either  in  sufficient  quantity,  and 
still  less  in  a  condition  of  sufficient  purity,  to  admit  of  such  a 
chemical  investigation  as  would  lead  to  the  determination  of  its 
composition.  But  by  analogy  with  the  known  relationship  of 
biliverdin  to  bilirubin,  and  from  the  evidence  afforded  by  the 
composition  of  choletelin  (see  below)  into  which  bilicyanin  may 
be  readily  converted  by  further  oxidation,  bilicyanin  will  probably 
be  found  to  differ  from  biliverdin  simply  by  the  addition  of  oxygen 
to  the  molecule  of  the  latter. 

Preparation.  Bilirubin  is  dissolved  in  chloroform  or  suspended 
in  alcohol  and  slowly  oxidised  either  by  gradual  addition  of  bro- 
mine or  fuming  nitric  acid ;  as  soon  as  the  mixture  is  of  a  bright 
blue  colour,  the  bilicyanin  is  precipitated  by  an  excess  of  water. 
As  thus  obtained  it  is  insoluble  in  water,  almost  insoluble  in  either 
ether  or  chloroform,  but  soluble  in  alcohol  and  alkalis.  In  pres- 
ence of  alkalis  it  is  still  almost  insoluble  in  either  ether  or  chloro- 
form ;  in  presence  of  acids  it  is  now  scarcely  soluble  in  water,  but 
soluble  in  ether  and  chloroform. 

Bilicyanin  is  for  practical  purposes  characterised  solely  by  its 
marked  absorption  spectrum.  This  consists  of  three  bands, —  one 
on  each  side  of  D,  that  to  the  red  side  of  D  being  the  darkest,  and 
one  between  h  and  F.  The  latter  is  probably  identical  with  the 
band  seen  in  acid  solutions  of  choletelin  and  due  to  the  produc- 
tion of  this  substance  in  small  quantity  during  the  oxidation  of 
bilirubin.  The  position  of  the  bands  varies  somewhat  according 
to  the  solvent  employed  and  as  to  whether  the  solution  is  acid  or 
alkaline. 

During  the  application  of  Gmelin's  test  for  bile-pigments  the  blue 
due  to  bilicyanin  is  bordered  by  a  violet  colour  and  this  by  a  red,  the 
final  and  permanent  colour  being  yellow.  Of  these  three  the  first  is 
not  as  yet  known  to  be  definitely  due  to  one  specific  substance ;  it  is 
most  probably  the  result  of  a  mixture  of  the  blue  of  bilicyanin  with 
the  red  of  the  next  product.  The  red  colour  is  on  the  other  hand 
supposedly  due  to  a  definite  pigment  sometimes  called  bilipurpurin, 
of  which  however  nothing  definite  is  as  yet  known.  The  yellow 
marks  the  final  formation  of  choletelin. 

4.  Choletelin.    CieHigN^Oe.  (?) 

This  is  the  final  product  of  the  oxidation  of  bile-pigments.  It 
is  readily  obtained  by  suspending  bilirubin  in  alcohol  and  oxidis- 

1  Heynsius  u.  Campbell,  Pfliiger's  Arch.  Bd.  iv.  (1871),  S.  526,  x.  1875,  S.  246, 
gives  literature  of  this  and  other  bile-pigments. 


246  HYDROBILIRUBIN. 

ing  it  by  passing  the  fumes  of  nitrous  acid  into  the  mixture.  As 
soon  as  the  play  of  colours  is  complete  and  the  solution  is  of  a 
pure  yellow  colour,  it  is  poured  into  a  large  excess  of  water,  from 
which  on  more  or  less  prolonged  standing  choletelin  separates  out 
as  a  flocculent  mass,  which  if  washed  and  dried  yields  a  brown 
powder.i  It  is  readily  soluble  in  alkalis,  as  also  in  either  alcohol, 
chloroform,  or  ether,  but  least  so  in  the  two  last  solvents.  None 
of  the  solutions  exhibit  any  fluorescence  even  after  the  addition 
of  zinc  chloride.  In  this  it  differs  markedly  from  urobilin,  a  weJl- 
known  yellow  urinary  pigment.  The  above  statements  scarcely 
provide  any  certain  means  of  identifying  choletelin  as  a  chemical 
substance,  and  no  specific  test  for  it  has  as  yet  been  described. 
Neither  is  it  quite  certainly  characterised  by  its  absorption  spec- 
trum, so  far  at  least  as  any  specific  bands  are  concerned.  Indeed 
there  has  been  very  great  difference  of  opinion  as  to  whether  it 
ever  gives  any  band  at  all,  and  if  it  does,  where  this  band  is  situ- 
ated. With  our  existing  knowledge  it  seems  safe  to  say  that  in 
alkaline  solutions  choletelin  shows  no  absorption  band,  and  that 
in  acid  solutions  a  band  may  be,  and  frequently  is  seen,  lying  be- 
tween h  and  F.  The  uncertainty  as  to  its  spectroscopic  properties 
led  some  of  the  older  observers  ^  to  regard  choletelin  as  identical 
with  hydrobilirubin  (urobilin).  This  view  is  however  quite  un- 
tenable both  as  the  result  of  purely  chemical  investigations  ^  and 
of  spectrophotometric  determinations  of  the  optical  properties  of 
the  two  substances.* 

5.     Hydrobilirubin.     CsaHioNiOy. 

When  bilirubin  is  dissolved  in  dilute  caustic  potash  or  soda  or 
suspended  in  water  and  treated  with  sodium-amalgam  in  succes- 
sive portions,  air  being  at  the  same  time  carefully  excluded,  it  is 
observed  that  at  first  no  hydrogen  is  evolved ;  the  dark-coloured 
solution  becomes  gradually  lighter  in  colour  and  more  transparent, 
until  at  the  end  of  two  or  three  days  it  is  bright  yellow  or  brown- 
ish-yellow, and  now  hydrogen  begins  to  come  off  from  the  mixture. 
At  this  stage  the  supernatant  fluid  should  be  poured  off  from  the 
metallic  mercury  which  has  accumulated,  and  if  it  is  now  acidu- 
lated strongly  with  either  hydrochloric  or  acetic  acid,  it  yields  a 
more  or  less  copious  flocculent  precipitate  of  a  dark  reddish-brown 
colour.  This  precipitate  is  impure  hydrobilirubin.  It  is  purified 
by  being  redissolved  in  ammonia,  reprecipitated  from  this  solution 
by  the  addition  of  acid,  and  finally  washed  with  water.     At  first 

1  Maly,  Siiz.  d.  h.  AJcad.  d.  Wiss.  Wien,  Bd.  lvii.  (1868),  2  Abth.  Feb.-Hft.  lix. 
(1869),  2  Abth.     Ap.-Hft. 

2  Heynsius  u.  Campbell,  loc.  cit.  Stokvis,  Centralb.  f.  d.  med.  Wiss.  1873,  S.  211, 
449. 

3  Maly,  Ihid.  S.  321,  and  more  particularly  Liebermann,  Ffliiger's  Arch.  Bd. 
XI.   (1875),  S.   181. 

*  Vierordt,  Zt.f.  Biol.  Bd.  n.  (1874),  S.  399. 


CHEMICAL  BASIS   OF  THE  ANIMAL   BODY.        247 

during  the  washing  a  considerable  amount  of  the  substance  passes 
into  solution,  but  as  the  merely  adherent  salts  are  washed  away, 
it  becomes  less  and  less  soluble  in  water  until  at  last  it  is  almost 
insoluble.  When  dried  it  takes  the  form  of  a  dai'k  reddish-brown 
amorphous  powder,  which  is  readily  soluble  in  alcohol  and  chloro- 
form, and  but  sparingly  soluble  in  pure  ether.  It  is  also  very 
soluble  in  alkaline  solutions,  to  which  it  imparts  a  yellow  colour 
as  of  normal  urine  :  when  acidulated  the  solutions  turn  red.^ 

The  acid  solutions  of  hydrobilirubin  show  a  marked  absorption 
band  between  h  and  F  which  becomes  fainter  if  ammonia  is  added 
until  the  reaction  is  alkaline.  But  on  the  subsequent  addition  of 
a  few  drops  of  a  solution  of  zinc  chloride,  the  band  reappears 
with  usually  increased  intensity,  though  shifted  slightly  towards 
the  violet  end  of  the  spectrum.^  This  alkaline  solution  to  which 
the  zinc  salt  has  been  added  also  shows,  in  marked  distinction 
to  the  acid  solutions,  a  brilliant  fluorescence  which  is  most  charac- 
teristic of  the  substance,  being  of  a  bright  rosy-red  colour  by 
transmitted,  and  bright  green  by  reflected  light. 

Previously  to  the  discovery  of  hydrobilirubin  by  Maly,  a  well- 
characterised  urinary  pigment  had  been  isolated  and  described  by 
Jaffe  under  the  name  of  urobilin  (see  below),  while  about  the 
same  time  that  Maly's  work  was  carried  on,  a  pigment  had  been 
obtained  from  fseces  and  described,  under  the  name  of  stercobilin, 
as  identical  with  urobilin.^  Careful  comparison  by  Maly  of  his 
hydrobilirubin  with  urobilin  led  him  to  assert  the  complete  iden- 
tity of  the  two  substances.  This  view  has  been  most  generally 
adopted,  and  is  probably  correct  as  a  broad  statement  of  facts. 
There  are  on  the  other  hand  several  observers  who  have  expressed 
themselves  against  the  exact  identity  of  these  substances.*  Their 
views  are  however  based  on  comparatively  slight  and  inconclusive 
spectroscopic  differences  between  the  natural  and  artificially  pre- 
pared substances  and  on  other  differences,  such  as  of  the  intensity 
of  their  fluorescent  activity,  which  are  still  less  conclusive.  For  the 
present  the  evidence  of  close  relationship  if  not  of  absolute  iden- 
tity suffices  fully  as  a  basis  for  our  belief  in  the  genetic  relation- 
ship of  the  bile  and  urinary  pigments  and  of  the  ultimate 
derivation  of  these  from  the  colouring-matter  of  the  blood. 

During  his  earlier  researches  on  the  pigments  of  blood  Hoppe- 
Seyler  described  a  product  resulting  from  the  reduction  of  haema- 
tin  in  acid  solution  by  the  action  of  zinc  and  hydrochloric  acid, 

1  Maly,  Centralh.f.  d.  med.  Wiss.  1871,  S.  849.  Annal.  d.  Chem.  Ed.  163  (1872) 
S.  77. 

2  Vierordt,  Zt.  f.  Biol.  Bd.  ix.  (1873),  S.  160.  See  later  'Quantitative  Spectral- 
analyse,'  1876,  S.  99. 

3"Vanlair  u.  Masius,  Centralh.f.  d.  med.  Wiss.  1871,  S.  369.  Cf.  Jaffe,  Ibid.  S. 
465. 

4  See  MacMunn,  Clinical  Chemistri/  of  Urine,  1889,  p.  105,  or  Jl.  of  Physiol. 
Vol.  X.  (1889),  p.  72.  Contains  all  necessary  references.  But  as  against  Disque 
see  also  Maly,  Pflijger's  Arch.  Bd.  xx.  (1879),  S.  331. 


248  OEIGIN  OF  BILE-PIGMENTS. 

characterised  by  one  absorption  band  between  h  and  F  and,  as  he 
then  said,  two  other  bands.  ^  After  the  appearance  of  Maly's  work 
he  was  led  to  suspect  that  the  substance  he  had  previously  de- 
scribed was  in  reality  identical  with  hydrobilirubin  and  therefore 
with  urobilin,  a  conclusion  which  he  verified  by  a  careful  repeti- 
tion of  his  earlier  experiments  .^ 

More  recently  Nencki  and  Sieber  have  prepared  a  similar  pigment 
by  the  action  of  hydrochloric  acid  and  zinc  on  their  hsematoporphyrin, 
to  which  latter  substance,  as  was  stated  above,  they  assigned  a  formula 
identical  with  that  of  bilirubin.  They  state  however  that  the  pigment 
(urobilin)  is  not  quite  identical  as  obtained  on  the  one  hand  by  the 
action  of  nascent  hydrogen  on  bilirubin,  and  on  the  other  hand  on  their 
hsematoporphyrin.  ^ 

Assuming  then  the  identity  of  these  substances  we  have  in 
Hoppe-Seyler's  work  the  best  and  most  direct  chemical  evidence 
of  the  relationship  between  the  colouring-matters  of  the  blood 
and  bile.  For  if  one  and  the  same  substance,  viz.  urobilin,  can 
be  prepared  by  the  same  means,  namely  reduction  (hydrogena- 
tion)  from  both  hsematin  (haemoglobin)  and  bilirubin,  these  two 
substances  must  be  themselves  closely  related.  It  has  not  how- 
ever as  yet  been  found  possible  to  produce  a  bile-pigment  directly 
from  haemoglobin  or  hsematin  by  any  artificial  process  outside  the 
animal  body.  The  derivation  of  the  urinary  pigments  (urobilin) 
from  those  of  bile  presents  no  difficulty  when  it  is  remembered 
that  a  not  inconsiderable  quantity  of  hydrogen  is  present  in  the 
gases  of  the  intestine  (§  282)  which  may  be  accounted  for  by 
(butyric)  fermentative  processes  (p.  105),  and  that  this  hydrogen 
might  in  its  nascent  state  readily  produce  the  simple  change 
which  is  known  to  occur  when  bilirubin  is  converted  into 
hydrobilirubin  or  urobilin.  And  here  it  is  interesting  to  note 
that  hydrobilirubin  is  readily  absorbed  and  excreted  in  the 
urine  either  when  placed  in  the  alimentary  canal  or  injected 
subcutaneously. 

The  question  of  pigmentary  relationships  to  which  reference 
has  just  been  made  suggest  the  present  as  a  convenient  place  to 
enter  into  further  details  on  the  now  undoubted  but  once  dis- 
puted derivation  of  the  bile-pigments  from  the  colouring-matter 
of  blood  (see  §  477). 

The  starting  point  for  this  view  was  the  discovery  and  descrip- 
tion of  haematoidin  crystals  by  Virchow  (see  p.  239)  as  occurring 
in  old  blood-clots  in  parts  of  the  body  remote  from  the  liver  and 
in  which  it  was  inconceivable  that  they  could  have  arisen  by  any 
process  other  than  a  gradual  formation  from  the  pigment  of  the 

1  Med.-chem.  Untersuch.  Hft.  4,  1871,  S.  536. 

2  Ber.  d.  d.  chem.  GeselL  Bd.  vii.  (1874),  S.  1065. 

3  Monatsh.  f.  Chem.  Bd.  ix.  (1888),  S.  115;  Arch.  f.  exp.  Path.  u.  PharmaJcol.  Bd. 
XXIV.  (1888),  S.  430. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        249 

red  corpuscles,  followed  as  this  was  by  proofs  of  the  identity  of 
hsematoidin  and  bilirubin.  This  was  followed  ^  by  experiments 
on  the  injection  of  bile-salts  into  the  blood  and  an  accompanyincr 
output  of  bile-pigments  in  the  urine,  to  which  the  true  signifi- 
cance was  subsequently  attached  by  Kiihne,  namely  that  the 
pigments  arose  from  a  conversion  of  h&emoglobin  set  free  from 
the  corpuscles  under  the  solvent  action  of  the  bile-salts.  This 
he  confirmed  by  injections  of  hsemoglobin  in  solution.^  These 
views  were  however  opposed  on  the  basis  of  similar  experiments 
in  which  it  was  stated  that  either  no  bile-pigments  appeared  in 
the  urine  as  the  result  of  injections  of  haemoglobin  into  the  vas- 
cular system,  or  that  if  they  did,  they  were  due  merely  to  an  ac- 
cumulation of  that  small  amount  which  is  frequently  present  in 
the  urine  of  dogs.^  But  the  careful  subsequent  experiments  of 
Tarchanoff,  in  which  he  endeavoured  to  avoid  many  obvious 
sources  of  error  present  in  those  of  Naunyn  and  Steiner,  are 
more  usually  regarded  as  having  afforded  definite  and  conclusive 
confirmation  of  the  earlier  views.^  This  observer  further  found 
a  considerably  increased  amount  of  bile-pigments  in  the  bile  col- 
lected during  the  experiments,  and  came  to  the  conclusion  that 
the  conversion  of  blood-  into  bile-pigments  takes  place  in  the 
blood-vessels,  a  part  being  excreted  in  the  urine,  while  the  larger 
part  passes  out  in  the  bile.  He  showed  in  confirmation  of  earlier 
experiments  ^  that  the  liver  is  extremely  active  in  excreting  bili- 
rubin injected  into  the  blood-vessels  ;  practically  the  whole  of  it 
passes  out  in  the  bile.^  The  relationships  thus  indicated  receive 
further  confirmation  from  the  observation  that  in  many  patho- 
logical conditions  of  the  horse,  bile-pigments  are  copiously  found 
in  its  tissues  and  transudations,  accompanied  by  blood-pigments, 
and  that  solutions  of  haemoglobin  when  injected  into  the  sub- 
cutaneous tissue  of  this  animal  become  after  a  few  days  partially 
converted  in  situ  into  granules  and  flakes  which  are  of  a  yellow 
or  orange  colour  and  yield  an  intense  Gmelin's  reaction." 
Finally  by  the  action  of  phenylhydrazin  on  hsematin  and  on 
bilirubin  products  are  obtained  which  in  each  case  exhibit  a 
similar  and  marked  play  of  colours  under  the  action  of  fuming 
(yellow)  nitric  acid.^ 


1  Freriehs  u.  Staedeler,  Miiller's  Arch.  Jahrsj.  1856,  S.  .55. 

2  Virchow's  Arch.  Bd.  xiv.  (1858),  S.  310.    ''Cf.  Phi/siol.  Chem.  1868,  S.  89. 

^  'Naunyn,  Arch.  f.  A7iat.  u.  Phi/siol.  Jahvg.  1868,' S.  401.  Steiner,  Ibid.  1873, 
S.  160.     Contain  full  references  to  all  then  existing  literature. 

4  Pfl tiger's  Arch.  Bd.  ix.  (1874),  Sn.  53,  329. 

5  Feltz  et  Ritter,  Jn.  de  I'Anat.  et  de  la  Physiol.  1870,  p.  315.  Cf.  Vossius,  Arch. 
f.  exp.  Path.  u.  Pharmahol.  Bd.  xi.  (1879),  S.  426. 

^  See  later  Stadelmann,  Ibid.  Bd.  xv.  (1882),  S.  237,  and  (in  connection  with  the 
next  reference)  Bd.  xxvii.  (1890),  S.  93. 

■^  Latschenberger,  Zt.  f.  Veterinarkunde,  Bd.  i.  (1886),  S.  47.  Monatsh.  f.  Chem. 
Bd.  IX.  (1888),  S.  52. 

8  Filehne,  Verhand.  d.  Congresses  f.  inn.  Med.  Wiesbaden,  Ref.  in  Centralb.  f. 
Min.  Med.  1888. 


250  OEIGIN   OF  BILE-PIGMENTS. 

One  point  still  remains  for  discussion.  It  has  been  seen  that 
bile-pigments  can  be  formed  from  those  of  the  blood  in  outlying 
parts  of  the  body  without  the  intervention  of  the  liver.  Are  we 
therefore  to  suppose  that  the  liver  is  similarly  inoperative  in  that 
increased  'formation  and  excretion  of  bile-pigments,  both  in  the 
urine  and  bile,  which  result  from  the  intravascular  injection  of 
hsemoglobin  ?  Opinions  have  differed  on  this  point.  It  is  on  the 
whole  more  probable  that  the  liver  is  in  all  cases  the  chief  factor 
in  the  conversion.  The  normal  production  of  bile-pigments  is 
entirely  due  to  hepatic  activity,  for  no  pigments  are  accumulated 
in  the  body  after  extirpation  of  the  liver  in  frogs  or  its  exclusion 
from  the  circulation  in  birds.^  This  accords  with  the  fact  that 
apparently  the  larger  part  of  the  pigments  resulting  from  the  in- 
jection of  haemoglobin  pass  out  in  the  bile  while  but  little  goes 
into  the  urine.  If  this  is  so,  how  shall  we  account  for  the  excre- 
tion of  the  latter  and  smaller  portion  by  the  kidneys  ?  It  is 
known  that  the  liver  is  peculiarly  liable  under  the  influence  of 
but  slight  operative  and  other  influences  to  pass  some  of  its  pro- 
ducts over  into  its  lymphatics  whence  they  make  their  way  into 
the  blood-vessels  and  may  hence  be  excreted  by  the  kidneys. 
Very  slight  obstruction  of  the  bile-duct  suffices  to  produce  this 
result,  and  it  has  been  observed  that  the  bile  formed  after  injec- 
tions of  haemoglobin  is  unusually  viscid.  The  views  here  put 
forward  (see  also  §  477)  are  further  in  complete  accord  with  the 
facts  that  hsematin  (hsemochromogen)  readily  loses  iron  and 
yields  hsematoporphyrin  03211301^405  which  differs  but  slightly  in 
composition  from  bilirubin  (Ci6lIi8N203)2,  and  that  it  is  precisely 
in  bile  and  very  largely  in  the  liver  that  we  meet  with  consider- 
able quantities  of  iron  in  some  as  yet  not  well-known  form.^ 
The  possible  function  of  the  spleen  as  an  organ  in  which  a  con- 
siderable disintegration  of  red  corpuscles  takes  place,  in  providing 
the  material  requisite  for  the  formation  of  bile-pigments  by  the 
liver  has  been  already  discussed  ^  (§  478). 

As  already  stated  herbivorous  bile,  as  of  ox  and  sheep,  frequently 
shows  absorption  bands  even  when  fresh.  These  are  regarded  by 
MacMunn  as  due  to  a  substance  to  which  he  has  given  the  name 
cholo-hsematin  since  it  occurs  in  bile  and.  as  the  action  of  sodium- 
amalgam  shows,  is  related  to  hsematin.  The  bands  more  usually  seen 
are  three,  two  near  D  and  one  near  E^ 

1  Stern,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  xix.  (1885),  S.  39.  Minkowski  u. 
Naunyn,  Ibid.  Bd.'xxi.  (1886),  S.  1. 

2  Zaleski,  Zt.  f.  physiol.  Chem.  Bd.  x.  (1886),  S.  453.  See  also  Virchow's  Arch. 
Bd.  CIV.  (1886),  S.  91. 

'  According  to  Schafer,  Proc.  Phj/siol.  Son.  1890,  No.  3  (see  Jl.  of  Physiol.  Vol. 
XI.),  there  is  no  evidence  of  any  discharge  of  hcemoglobin  from  the  spleen  in  the 
blood  of  the  vein  of  this  organ. 

*  JL  of  Physiol.  Vol.  vi.  (1884),  p.  24. 


CHEMICAL  BASIS   OF  THE  AJ^IMAL  BODY.       251 


THE   PIGMENTS   OF   UEINE.i 

When  fresh,  normal  urines  are  examined  spectrophotometrically 
it  is  found  that  the  extinction  coefficients  (see  p.  225)  for  any 
given  portion  of  the  spectrum  of  the  several  fluids  do  not  bear  a 
constant  ratio  each  to  the  other.  If  the  urines  contained  only 
one  colouring-substance,  then  no  matter  how  much  the  absolute 
value  of  the  extinction  coefficients  varied  for  different  regions  of 
the  spectrum,  their  ratios  would  be  constant  for  any  given  region. 
From  this  it  appears  probable  at  the  outset  that  even  normal 
urine  is  coloured  by  at  least  two  if  not  more  pigments.^  Our 
knowledge  of  these  pigments  is  at  present  imperfect  and  almost 
limited  to  that  of  one  substance,  namely  urobilin,  and  even  with 
respect  to  this  one,  considerable  difference  of  opinion  exists  as  to 
its  nature  and  relationships  to  the  other  pigments  of  the  body 
from  which  it  is  supposed  to  be  ultimately  derived.  The  reasons 
for  this  are  simple.  It  is  extremely  probable  that  normal  urine 
is  often  coloured  by  some  chromogenic  mother-substance  (cf.  zy- 
mogens) rather  than  by  the  fully  formed  pigment.  In  the  next 
place,  since  the  colouring-matters  are  normally  present  in  but 
very  small  amount,  and  since  they  are  not  known  to  be  crystal- 
lisable  or  to  form  definite  compounds  with  well-known  precipi- 
tants,  they  have  not  as  yet,  with  the  exception  perhaps  of 
urobilin,  been  obtained  either  with  any  guarantee  of  their  purity 
or  in  quantities  sufficient  to  admit  of  ultimate  analysis.  Hence 
our  knowledge  of  them  is  chiefly  based  upon  their  spectroscopic 
properties.  They  are  further  most  probably  far  from  stable  sub- 
stances, so  that  they  may  undergo  some  considerable  change 
either  by  mere  exposure  to  the  air  (oxygen)  or  as  the  result  of 
the  various  and  often  different  methods  of  extraction  and  prepara- 
tion employed  by  various  authors.  This,  together  with  the  fact 
that  the  position  of  the  absorption  bands  may  vary  somewhat 
with  the  reaction  of  the  solution  and  the  nature  of  the  solvent, 
&c.,  accounts  with  but  little  doubt  not  only  for  the  extremely  nu- 
merous and  insufficiently  characterised  pigments  which  have  at 
one  time  or  another  been  obtained  from  urine,  but  also  for  much 
of  the  conflict  and  confusion  of  opinion  which  exists  as  to  the  na- 
ture and  relationships  of  those  pigments  of  which  we  can  speak 
with  most  confidence. 

1.    Urobilin.    C32H40N4O7.  (?) 

This,  the  best  known  and  most  definitely  characterised  of  the 
urinary  pigments,  was  first  described  by  Jafi"d  who  regarded  it  as 

1  For  references  to  the  principal  earlier  works  on  urinary  pigments  see  Udranszky, 
Zt.  f.  physiol.  Cliem.  Bd.  xi.  (1887),  S.  537,  and  for  all  details  consult  Neubaner  u. 
Vogel,  Analyse  des  Harms,  Aufl.  ix.  1890. 

'^  Vierordt,  Die  quantit.  Spectralanalyse,  1876,  S.  78. 


252  UKOBILIN. 

the  chief  colouring-substance  of  normal  urine,  while  present  in 
much  larger  amounts  in  the  urine  of  fever.  ^  He  also  obtained  it 
occasionally  from  bile,  the  name  urobilin  thus  nidicatmg  its  double 
source.  In  fresh  normal  urine  the  amount  was  frequently  ex- 
tremely small,  but  was  observed  to  increase  on  standing  exposed 
to  the  air  (oxygen),  a  result  due  to  the  probable  presence  in  the 
urine  of  some  chromogen  or  mother-substance  (urobilinogen)^  of 
the  urobilin.  The  amount  of  this  pigment  in  urine  is  too  small 
to  provide  adequate  material  for  an  elementary  analysis,  so  that  it 
was  at  first  characterised  by  its  solubilities  in  various  fluids,  by 
the  strongly-marked  fluorescence  of  certain  of  these  solutions 
and  more  particularly  by  the  absorption-spectrum  it  exhibited. 
The  subsequent  preparation  of  hydrobilirubin  from  bilirubin,  and 
the  establishment  of  its  identity  with  urobilin  (p.  246)  provided 
for  the  first  time  a  mass  of  the  substance  sufficient  to  admit  of 
analysis,  and  upon  this  the  formula  given  above  for  urobilin  is 
based.  It  must  not  however  be  forgotten  that  the  identity  of 
the  two  pigments  is  disputed  by  several  observers,  although  the 
balance  of  belief  seems  as  yet  to  support  it.  It  will  conduce  to 
clearness  if  we  incline  for  the  present  to  this  belief  and  describe 
the  preparation  and  properties  of  urobilin  as  given  by  Jaff'i^,  on 
the  assumption  that  it  is  identical  with  hydrobilirubin,  and  then 
subsequently  give  a  short  account  of  the  opposing  views. 

Prepai'ation  from  urine.  Several  methods  may  be  adopted  ;  of 
these  only  the  broader  facts  can  here  be  given,  but  they  suffice 
to  provide  solutions  which  exhibit  the  characteristic  spectra. 
(i)  When  urine  contained  much  urobilin  Jaffd  precipitated  it  by 
the  addition  of  chloride  of  zinc  in  presence  of  an  excess  of  am- 
monia ;  if  but  little,  then  by  the  addition  of  basic  lead  acetate. 
These  precipitates  were  then  worked  up  by  processes  which  do 
not'  admit  of  a  suitably  brief  description.^  (ii)  Precipitate  the 
urine  completely  by  the  addition  first  of  normal  lead  acetate,  then 
of  the  basic  acetate.  Wash  the  precipitates,  dry  at  low  tempera- 
ture, and  extract  with  absolute  alcohol  (not  methylated  spirit) 
acidulated  with  1  —  2  p.c.  of  sulphuric  acid.  This  extract  may 
be  then  diluted  with  water  and  the  pigment  extracted  by  shaking 
up  with  chloroform,  in  which  it  is  readily  soluble.*  (iii)  The 
urine  is  acidulated  with  0*2  p.c.  of  sulphuric  acid  and  then  satu- 
rated with  neutral  ammonium  sulphate.  The  precipitate  thus 
obtained  is  then  collected  on  a  filter,  washed  with  an  acidulated 
saturated  solution  of  the  ammonium  salt,  freed  by  pressure  from 
adhering  fluid,  and  dissolved  by  gentle  warming  in  absolute  alcohol 

1  Ceniralb.  f.  d.  med.  Wiss.  1868,  S.  243 ;  1869,  S.  177.  Virchow's  Arch.  Bd. 
XLVii.  (1869),  S.  405. 

2  For  further  references  see  Neubauer  u.  Vogel,  Anal.  d.  Hams.  1890,  Sn.  331, 
336. 

^  For  details  see  Neubauer  u.  Vogel,  loc.  cit.  S.  334. 

*  Mac  Munn,  Proc.  Roy.  Soc.  pp.  26,  206.     Jl.  of  Physiol.  Vol.  x.  (1889),  p.  71. 


CHEMICAL  BASIS   OF  THE  ANIMAL  BODY.        253 

to  which  if  necessary  a  few  drops  of  ammonia  have  been  added.^ 
(ivj  Frequently  from  normal  urine,  the  more  readily  if  that  be 
highly  coloured,  a  solution  of  urobilin  may  be  obtained  by  simple 
agitation  with  chloroform,  or  by  gently  shaking  it  up  with  half  its 
volume  of  2^uTe  ether  free  from  all  traces  of  alcohol.  The  ether 
is  then  removed  by  a  separating  funnel,  evaporated  at  ordinary 
temperatures,  and  the  residue  dissolved  in  a  small  quantity  of 
absolute  alcohol.^ 

If  the  alcoholic  or  chloroformic  solutions  above  described  are 
evaporated  to  dryness  at  a  low  temperature,  the  urobilin  remains 
as  a  yellowish-brown  amorphous  pigment,  which  is  practically 
insoluble  in  water  except  in  presence  of  small  amounts  of  neutral 
salts,  very  slightly  soluble  in  either  ether  or  benzol,  readily  soluble 
in  alcohol  and  in  chloroform.  The  neutral  alcoholic  solutions  if 
dilute  are  yellow  with  a  rosy  tint,  and  if  strong  show  a  green 
fluorescence.  The  acid  solutions  are  reddish-yellow,  or  if  dilute 
bright  rose-coloured  and  do  not  fluoresce.  Alkaline  (alcoholic) 
solutions  are  yellow  or  yellowish-green  according  to  the  concen- 
tration and  usually  show  a  marked  fluorescence,  which  is  much 
increased  on  the  addition  of  a  solution  of  zinc  chloride,  appear- 
ing now  rose-coloured  by  transmitted  light  and  brilliant  green 
by  reflected. 

Spectra  of  urobilin.  Neutral  or  alkaline  alcoholic  solutions 
show  one  absorption  band  between  h  and  F.  In  alkaline  solution 
the  band  is  frequently  very  faint,  but  is  more  strongly  marked 
after  the  addition  of  zinc  chloride,  so  much  so  that  it  can  often 
only  be  distinctly  seen  after  the  addition  of  this  salt.  In  acid 
solutions  a  similar  band  is  seen,  situated  however  in  this  case 
slightly  more  towards  the  violet  end  of  the  spectrum. 

The  methods  given  above  for  the  preparation  of  urobilin,  indi- 
cate sufficiently  the  procedure  requisite  for  its  detection  in  solu- 
tions. As  already  stated  (p.  246)  the  position  of  the  absorption 
band  of  urobilin  is  very  similarly  situated  to  that  of  choletelin 
under  certain  conditions.  The  conflict  of  opinion  as  to  the  identity 
of  the  two  substances  has  been  dealt  with  above. 

It  now  remains  to  give  a  short  account  of  the  more  recent  views 
on  urobilin  and  its  relationship  to  other  pigmentary  substances  to 
which  reference  has  already  been  made.^ 

Mac  Munn  distinguishes  between  two  forms  of  urobilin,  viz. 
normal  and  febrile  or  pathological.  They  are  both  obtained  from 
urine  by  the  same   method    (see    above)    and  differ  as  follows. 

1  Mehu,  Jowrn.  d.  pharm.  et  de  chim.  T.  xxviii.  (1878),  p.  159.  This  method  is 
good,  as  avoiding  to  a  considerable  extent  any  alteration  of  the  pigments  by  the 
process  employed. 

2  E.  Salkowski,  Zt.f.  physiol  Chem.  Bd.  iv.  (1880),  S.  134. 

3  Mac  Munn,  Clinical  Chem.  of  Urine,  1889,  p.  104.  Gives  all  necessary  references. 
For  spectra  see  Jl.  of  Physiol.  Vol.  x.  (1889),  p.  116. 


254  UROBILIN. 

(i)  Normal  urohilin.  In  acid  alcoholic  solution  it  shows  one 
absorption  band,  close  to  and  enclosing  F:  this  band  disappears 
when  the  solution  is  neutralised  by  alkalis.  If  treated  with  zinc 
chloride  in  presence  of  ammonia  this  band  is  replaced  by  one 
narrower  and  nearer  the  red  end  of  the  spectrum,  while  at  the 
same  time  a  green  fluorescence  is  observed,  but  much  less  marked 
than  in  the  case  of  febrile  urobilin,  (ii)  Febrile  itrolilin.  The 
solubilities  of  this  substance  are  the  same  as  of  the  preceding 
form.  On  the  other  hand  the  band  at  F  is  broader  and  darker 
than  is  that  of  normal  urobilin,  and  further  in  an  ethereal  acid 
solution  two  other  bands  may  be  seen,  one  adjoining  D  towards 
the  red,  the  other  mid-way  between  D  and  E.  These  last  two 
bands  are  invisible  in  urine.  By  prolonged  action  of  sodium 
amalgam  on  an  alcoholic  solution  of  normal  urobilin,  fibrile  urobi- 
lin  is  obtained.  The  spectrum  of  normal  urobilin  is  the  same  as 
that  of  choletelin,  but  the  substances  differ  with  respect  to  the 
greater  ease  with  which  choletelin  may  be  reduced  to  febrile 
urobilin.  Normal  urobilin  is  regarded  as  differing  from  hydro- 
bilirubin,  the  evidence  being  deduced  from  spectroscopic  obser- 
vations. Febrile  urobilin  on  the  other  hand  is  identical  with 
stercobilin  and  is  apparently  the  pigment  to  which  the  absorption 
spectra  of  the  bile  of  some  animals  is  due.^ 

In  concluding  this  account  of  urobilin  and  allied  substances  it 
may  be  well  once  more  to  draw  attention  to  the  fact  that  the 
differences  of  opinion  among  the  various  observers  is  based  almost 
entirely  on  spectroscopic  appearances.  These  are  far  from  conclu- 
sive for  there  is  no  guarantee  that  in  any  given  case  the  solution 
under  examination  contains  only  one  pigment.  It  may  contain  at 
most  a  preponderance  of  this  one  but  frequently  mixed  with  other 
pigments  which  are  derived  either  from  the  fluid  originally  oper- 
ated upon,  or  are  decomposition  products  resulting  from  the  action 
of  the  reagents  employed.^  The  final  solution  of  the  questions 
raised  above  will  only  be  supplied  by  a  purely  chemical  investiga- 
tion of  the  several  substances  under  discussion ;  such  an  investi- 
gation would  however  be  one  of  extreme  difficulty. 

Thudichum  considered  that  normal  urine  contains  only  one  pigment, 
which  he  called  urochrome.^  Maly  regarded  this  as  the  same  as  urobi- 
lin.'*    More  recently  Thudichum  has  upheld  his  former  views. ^ 

1  Mac  Munn,  The  Spectroscope  in  Med.  1880,  p.  156.  A  tabular  conspect  of  the 
above  statements  is  given  by  Halliburton,  Chem.  Physiol,  and  Pathol.  1891,  p.  752. 

-  Thus  Vierordt  has  shown  that  if  the  urinary  pigments  are  precipitated  by  the 
acetates  of  lead  and  extracted  from  this  by  absolute  alcohol  acidulated  with  oxalic 
acid,  the  coloured  solution  thus  obtained  possesses  optical  properties  quite  different 
from  those  of  the  original  urine ;  a  result  which  indicates  that  the  pigments  have 
been  considerably  changed  during  extraction.  Die  quantitat.  Spectrcdanal.ijse,  1876, 
S.  96.  •  ' 

3  Brit.  Med.  Jl.  No.  201,  1864,  p.  509. 

*  Liebig's  Ann.  Bd.  clxiii.  (1872),  S.  90. 

5  Jl.  Chem.  Soc.  Ser.  2,  Vol.  xiii.  (1875),  pp.  397,  401.  _    ... 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.        255 


2.  Uroerythrin. 

This  is  a  pigment  of  which  but  little  is  known.  It  is  regarded 
as  the  colouring-substance  of  certain  bright-red  (pink)  urinary 
deposits  and  as  possibly  occurring  in  the  highly  coloured  urines 
of  rheumatism,  &c.  It  appears  to  be  an  amorphous  reddish  sub- 
stance, with  an  acid  reaction,  slowly  soluble  in  either  water, 
alcohol,  or  ether.^  Treated  with  caustic  alkalis  it  turns  green, 
more  particularly  when  in  the  solid  form.  In  alcoholic  solution 
obtained  by  boiling  pink  urates  with  alcohol  it  shows  two  ill- 
defined  absorption  bands  between  D  and  F!^ 

3.  Urohcematoporphyrin. 

This  pigment  was  first  described  by  Mac  Munn  (under  the  name 
of  urohsematin)  as  occasionally  occurring  in  certain  pathological 
urines  as  of  acute  rheumatism,  Addison's  disease,  &c.  and  to  it  he 
gave  the  present  name  from  certain  resemblances  of  its  spectra  to 
those  of  hsematoporphyrin.^  It  is  obtained  from  urine  by  the 
method  employed  for  the  separation  of  urobilin,  or  artificially  by 
the  action  of  reducing  agents  on  hsematin,  this  being  the  supposed 
source  of  its  origin  in  the  body.  It  is  soluble  in  either  alcohol, 
ether,  benzol,  or  chloroform.  In  acid  alcoholic  solution  it  shows 
three  absorption  bands,  one  narrow  adjoining  D  on  the  red  side  of 
this  line,  one  half  way  between  D  and  E,  and  one  between  h  and 
F  closely  resembling  the  band  of  urobilin.  There  is  also  occa- 
sionally a  fourth  very  faint  band  between  the  first  two  bands 
described  above.  In  alcoholic  solution  made  alkaline  by  ammonia 
it  yields  a  spectrum  closely  resembling  that  of  hsematoporphyrin 
(see  above  p.  238).  But  unlike  the  latter  substance  its  solutions 
show  a  very  faint  green  fluorescence  on  the  addition  of  zinc 
chloride  and  ammonia.  The  occurrence  of  hsematoporphyrin  in 
urine  has  been  frequently  recorded*  and  from  the  spectroscopic 
appearances  described  above,  some  observers  are  inclined  to  the 
view  that  urohsematoporphyrin  is  not  a  single  substance  but  a 
mixture  of  hsematoporphyrin  with  some  pigment  closely  resem- 
bling urobilin 

Urohsematoporphyrin  is  perhaps  closely  related  to  two  pigments 
known  as  urorubrolifematin  and  urofuscohiBmatin  obtained  from  a 
case  of  leprosy  ^  (Mac  Munn) . 

1  Heller,  in  his  Archiv.  (2)  Bd.  in.  (1854),  S.  361. 

2  Mac  Munn,  Proc.  Roy.  Soc.  Vol.  xxxv.  (1883),  pp.  132,  370. 

3  Jl.  of  Physiol.  Vols.  vi.  (1884),  p.  36  ;   x.  (1889),  p.  73. 

*  See  most  recently  E.  Salkowski,  Zt.  f.  physiol.  Chem.  Bd.  xv.  (1891),  S.  286. 
There  was  in  the  cases  examined  some  evidence  that  the  occurrence  of  h^mato- 
porphyrin  iu  the  urine  was  perhaps  not  unconnected  with  the  administration  of 
sulphonal. 

5  Baumstark,  Ber.  d.  d.  chem.  Gesdl.  Bd.  vii.  (1874),  S.  1170.  Pfliiger's  Arch. 
Bd.  IX.  (1874),  S.  568.     Cf.  Hoppe-Seyler,  Physiol.  Chem.  1879,  S.  875. 


256         HUMUS   PIGMENTS.     UEINAEY  MELANIN. 

4.  Humus  pigments. 

When  carbohydrates  are  treated  with  acids  or  alkalis,  among 
the  numerous  products  which  arise  are  certain  pigmentary  bodies 
of  a  more  or  less  dark-brown  colour.  A  similar  colouration  is 
well  known  as  occurring  in  fruits  when  bruised  or  exposed  to 
the  air,i  and  generally  in  decaying  vegetable  tissues.  These  sub- 
stances are  known  under  the  name  of  'humus.'  When  urine  is 
treated  with  acids  in  presence  of  oxygen  it  acquires  a  markedly 
darker  colour,  and  since  carbohydrates  in  small  amount  are  prob- 
ably present  in  all  urines,^  there  is  at  once  a  possibility  that  some 
at  least  of  the  observed  colouration  is  due  to  the  production  of 
humus-pigmented  substances  by  the  action  of  the  acids  on  the 
carbohydrates.  In  accordance  with  this  view  certain  so-called 
humus  pigments  have  been  prepared  from  urine,  but  our  knowl- 
edge of  them  is  as  yet  very  incomplete.  They  are  stated  to  be 
practically  insoluble  in  any  solvents  other  than  amyl-alcohol, 
strong  ammonia,  and  caustic  alkalis  :  the  solutions  show  no 
absorption  bands  when  examined  spectroscopically.  They  are 
further  said  to  account  for  the  usually  dark  colour  of  normal 
herbivorous  urine  and  of  urine  after  the  cutaneous  absorption 
of  carbolic  acid  and  several  other  aromatic  compounds.^ 

It  is  very  probable  that  several  dark-coloured  pigments  such  as  the 
uromelaniiis  of  Plosz  and  Thndicluira  obtained  by  the  action  of  acids 
on  urinary  pigments  or  chromogens  are  allied  to  if  not  identical  with 
these  humus  substances. 

5.  Urinary  melanin.* 

Certain  tumours  are  not  infrequently  observed  which  from 
their  extremely  dark  pigmentation  are  spoken  of  as  '  melanotic,' 
the  colouring-substance  being  known  as  melanin.^  The  urine  of 
patients  suffering  from  these  tumours  is  either  dark-brown  or 
black  when  voided,  or  speedily  assumes  this  colour  after  brief 
exposure  to  the  air  or  by  the  action  of  nitric  acid  or  other  oxidis- 
ing agents,  the  pigment  to  which  the  colour  is  due  being  ap- 
parently identical  with  that  present  in  the  tumour.  This  action 
of  oxidising  agents  indicates  that  here  also,  as  in  the  case  of 
other  urinary  pigments,  there  is  primarily  some  chromogenic 
forerunner  (melanogen)  of  the  actual  pigment.     This  chromogen 

1  Hoppe-Seyler,  Zt.  f.  physiol.  Chem.  Bd.  xiii.  (1889),  S.  66. 

2  SeeWedenski,  Ibid.  S.'l22.     E.  Salkowski,  Ibid.  S.  270. 

3  Udranszky,  Ibid.  Bde.  xi.  (1887),  S.  537,  xii.  (1888),  S.  33.  Contains  very  full 
references  to  other  works. 

*■  Morner,  Zt.  f.  physiol.  Chem.  Bde.  xi.  (1887),  S.  66,  xii.  (1888),  S.  229.  Gives 
list  of  literature  to  date.  See  also  Zeller,  Langenbeck's  Arch.  Bd.  xxix.  (1884),  S.  2, 
and  later  Brandl  u.  Pfeiffer,  Zeitsch.f.  Biol.  Bd.  xxvi.  (1890),  S.  348. 

^  The  name  melanin  is  more  usually  applied  as  a  generic  title  for  the  dark- 
brown  or  black  pigments,  such  as  occur  in  the  hair,  epidermis,  retinal  epithelium, 
choroid,  &c. 


CHEMICAL  BASIS   OF   THE  ANIMAL   BODY.        257 

may  be  partially  precipitated  from  the  urine  by  baryta  water  and 
completely  by  normal  lead  acetate.  When  the  latter  precipitate 
is  suspended  in  water  and  decomposed  by  sulphuretted  hydrogen, 
it  yields  a  colourless  solution  which  when  evaporated  to  dryness 
leaves  a  dark  amorphous  residue  insoluble  in  water,  ether,  cold 
alcohol,  acetic  acid,  and  dilute  mineral  acids.  The  fully  formed 
pigment  may,  like  its  chromogenic  forerunner,  be  partially  pre- 
cipitated by  baryta  water,  the  remainder  being  precipitable  by  the 
subsequent  addition  of  normal  lead  acetate.  The  baryta  precipi- 
tate contains  the  larger  amount  of  the  pigment,  and  from  it  the 
colouring-matter  may  be  more  easily  obtained  than  from  the 
precipitate  with  the  lead  salt,  since  the  latter  carries  down  other 
urinary  pigments  at  the  same  time.  The  isolation  of  the  urinary 
melanin  in  a  pure  form  from  the  baryta  compound  admits  of  no 
suitably  concise  description  ;  it  must  suffice  here  to  state  that  an 
impure  product  is  obtained  by  decomposing  the  compound  with 
sodium  carbonate  assisted  by  gentle  warmth  and  precipitating  the 
pigment  from  the  resulting  solution  by  a  slight  excess  of  sul- 
phuric acid.  The  product  when  purified  is  partly  insoluble, 
partly  soluble  in  acetic  acid  of  50 — 75  p.  c.  Of  these  portions 
the  former  when  dried  is  a  brownish-black  amorphous  powder, 
insoluble  in  either  water,  alcohol,  ether,  chloroform,  or  dilute 
(mineral)  acids,  but  readily  soluble  in  alkalis.  The  latter  was 
obtained  in  too  small  amounts  to  admit  of  complete  investigation. 
On  analysis  the  pigment  was  found  to  contain  iron  ('2  p.  c.) 
and  a  considerable  amount  of  sulphur  (9  p.  c.)  and  not  to 
show  any  absorption  bands  when  its  solutions  were  examined 
spectroscopically. 

This  pigment  appears  to  be  identical  with  one  previously  described 
under  the  name  of  phymatorhusin  as  obtained  from  melanotic  tumours, 
and  closelj^  allied  to  hyppomelanin  obtained  from  similar  tumours  of 
the  horse.  ^ 

When  melanotic  urines  are  treated  with  solutions  of  ferric 
chloride,  they  yield,  according  to  the  concentration  of  the  re- 
agent, either  a  dark -brown  cloudiness  or  else  a  black  precipitate 
soluble  in  excess  of  the  precipitant :  this  test  is  both  delicate  and 
characteristic.  Further  when  to  these  urines  a  dilute  solution  of 
sodium  nitroprusside  and  some  caustic  potash  is  added  they  fre- 
quently show  a  pink  or  red  colouration  which  turns  blue  on  the 
addition  of  acids,  owing  to  the  formation  of  Prussian  blue.  The 
latter  reaction  is  not  due  to  the  melanotic  pigment  but  to  some 
other  substance  simultaneously  excreted.^ 

1  Berdez  u.  Nencki,  Arch.  f.  exp.  Pathol,  u.  Pharmakol.  Bd.  xx.  (1886),  S.  346. 
Nencki  u.  Sieber,  Ibid.  Bd.  xxiv.  (1888),  S.  17.  See  also  Miura,  Virchow's  Arch. 
Bd.  CYii.  (1887),  S.  250. 

2  V.  Jaksch,  Zt.f.  physiol.  Chem.  Bd.  xiii.  (1889),  S.  385. 

17 


258      INDOXYL-PIGMENTS.     SKATOXYL-PIGMENTS. 

6.  Indoxyl-pigments. 

Of  the  total  indol  formed  in  the  alimentary  canal,  a  portion  is 
"excreted  with  the  faeces,  while  the  remainder  is  absorbed  and  re- 
appears in  the  urine  united  with  potassium  as  ethereal  compounds 
of  indoxyl  with  either  glycuronic  acid  (p.  107)  or  sulphuric  acid 
(p.  199),  the  latter  being  known  as  urinary  indican.  When 
warmed  with  hydrochloric  acid  these  compounds  are  decomposed, 
yielding  indoxyl  and  the  potassium  salt  of  the  corresponding  acid. 
If  the  decomposition  is  effected  in  the  absence  of  oxygen,  the  in- 
doxyl may  be  in  part  gradually  changed  into  an  amorphous  red- 
dish substance,  indigo-red,  which  is  insoluble  in  water,  but  yields 
a  red  solution  when  dissolved  in  alcohol,  ether,  or  chloroform,  i 
These  solutions  show  no  certainly  characteristic  absorption  bands. 
In  presence  of  oxygen  and  with  most  certainty  by  the  action  of 
an  oxidising  agent,  the  indoxyl  is  readily  converted  into  indigo- 
blue,  whose  properties  and  solubilities  have  been  already  suffi- 
ciently described.  Dilute  solutions  of  indigo-blue  exhibit  in  thin 
layers  one  absorption  band  in  the  red  lying  between  a  and  ^25 
C  ;  if  the  thickness  of  the  solution  be  increased  this  band  widens 
out  towards  D  and  at  the  same  time  a  second  faint  band  makes 
its  appearance  in  the  green  lying  between  D  50  ^  and  D  11  EP- 

The  numbers  just  given  refer  to  the  method  (Vierordt's)  frequently 
used  for  indicating  the  position  of  an  absorption  band.  In  this  the 
distance  between  any  two  of  the  fixed  lines  of  the  solar  spectrum  is  re- 
garded as  being  divided  into  100  equal  parts  and  the  extent  of  the 
band  is  given  by  reference  to  these  divisions.  Thus  if  a  band  is  de- 
scribed as  lying  between  D  oO  E  and  D  11  E  \i  implies  that  the  band 
begins  half  way  {■f'-^Q  of  the  distance)  between  D  and  E  and  extends 
to  yYo  of  the  distance  between  the  same  two  lines.®  (See  also  above, 
note  1,  p.  228.) 

Variable  accounts  of  the  above  pigments  may  be  obtained  from 
urines  during  their  spontaneous  decomposition  or  when  treated 
with  hydrochloric  acid  or  oxidising  agents,  the  amount  being 
greatest  in  herbivorous  urine  and  especially  great  in  certain 
pathological  urines  (see  p.  199).  They  have  also  been  met 
with  in  urinary  sediments  and  calculi.* 

7.  Skatoxyl-pigments. 

The  skatol  formed  in  the  alimentary  canal  gives  rise,  like  in- 
dol, to  compounds  of  skatoxyl  with  either  sulphuric  acid  or  glycu- 

1  Cf.  Nencki,  Ber.  d.  d.  chem.  Gesell.  Bd.  ix.  (1876),  S.  299,  and  see  MacMunn, 
Proc.  Roy.  Soc.  Vol.  xxxv.  (1883),  p.  370. 

2  Vierordt,  Zt.f.  Biol.  Bde  x.  (1874),  S.  27,  xi.  (1875),  S.  192. 

8  A  table  for  the  conversion  of  these  data  into  wave-length  limits  is  given  by  G. 
U.  H.  Kriiss,  Kolorimetrie  u.  quant.  Spektral analyse,  1891,  S.  290.  , 

*  Ord,  Berl.  klin.  Wochensch.  1878,  S.  365.  Chiari,  Prager  med.  Wochensck. 
1888,  S.  541. 


CHEMICAL  BASIS   OF  THE   ANIMAL  BODY.        259 

ronic  acid  (see  p.  202).  These  compounds  when  decomposed  by 
hydrochloric  acid  or  oxidising  agents  give  rise  to  a  colouring-mat- 
ter which  is  more  or  less  red  and  may  exhibit  a  distinct  and 
strong  purple  tint.^  The  pigment  is  insoluble  in  water,  but  solu- 
ble in  either  alcohol  or  chloroform,  also  when  freshly  prepared  in 
ether  but  less  so  if  it  has  been  kept  some  time.  Alcoholic  solu- 
tions are  of  a  reddish-violet  colour ;  ethereal  solutions  may  show 
a  green  fluorescence,  which  on  exposure  to  the  air  takes  on  a 
reddish  tinge.  It  is  also  soluble  in  hydrochloric  and  sulphuric 
acids,  giving  bright  red  or  pink  solutions,  and  in  alkalis  yield- 
ing yellow  solutions.  No  absorption  bands  for  this  substance 
have  as  yet  been  described  and  the  whole  subject  requires  further 
investigation. 

A  considerable  number  of  red  or  reddish-purple  pigments  have 
at  different  times  been  obtained  and  described  under  specific 
names  as  derived  either  from  pathological  urines  when  first 
voided,  or  from  the  spontaneous  decompositions  of  or  action  of 
mineral  acids  on  different  urines.  The  remarks  which  have  been 
made  on  the  indoxyl  and  skatoxyl  pigments  indicate  a  possibility 
that  they  may  all  have  a  common  origin  and  thus  be  closely  re- 
lated if  not  in  many  cases  identical.  In  the  absence  of  any  guar- 
antee of  the  purity  of  the  several  coloured  products  and  of  their 
not  having  undergone  some  change  during  the  operations  in- 
volved in  their  preparation,  no  authoritative  statement  on  this 
point  can  as  yet  be  made.  Indeed  the  whole  subject  of  the 
origin,  nature,  and  relationships  of  urinary  pigments  is  at  pres- 
ent in  a  state  of  considerable  confusion  and  uncertainty.^ 

The  urinary  pigments  so  far  dealt  with  may  be  regarded  as 
either  normal  or  pathological,  or  as  resulting  from  the  spontaneous 
or  artificial  decomposition  of  urinary  constituents  which  are  at  the 
outset  colourless.  In  addition  to  these,  other  colouring  substances 
are  not  infrequently  observed,  or  colour-reactions  obtained,  in 
urines  passed  after  the  administration  of  certain  drugs  or  the 
consumption  of  certain  vegetable  tissues.  They  are  in  many  cases 
not  unimportant  as  leading  at  first  sight  to  possibly  erroneous 
conclusions  as  to  the  presence  in  urine  of  pathologically  important 
pigments^  e.g.  of  bile  or  blood.  After  the  administration  of  rhu- 
barb or  senna,  the  urine  may  be  yellow  or  greenish-yellow,  due  to 
the  presence  of  chrysophanic  acid  [Ci^Hs  (CH3)  (0H)2  O2],  and 
similarly  after  the  use  of  santonin  (CisHigOg).  In  such  cases  if 
the  urine  is  strongly  alkaline  it  may  be  of  a  red  colour ;  this  is 
changed  to  yellow  on  the  addition  of  hydrochloric  acid,  and  if  it 

1  Otto,  Piluger's  Arch.  Bd.  xxxiii.  (1884),  S.  613.  Mester,  Zt.  f.  physioL  Chem. 
Bd.  XII.  (1888),  S.  130. 

2  For  further  literature  of  these  red  pigments  see  Mester,  he.  cit.  S.  143.  Also 
Bed.  Hin.  Wochensch.  1889,  Sn.  5,  202,  490,  520,9.53;  1890,  S.  58.5.  Centralb.  f. 
klin.  Med.  1889,  S.  505.     Stokvis  (Dutch),  Abst.  in  Maly's  Bericht.  1889,  S.  462. 


260  EETINAL  PIGMEiTTS. 

is  initially  acid,  it  turns  red  on  the  addition  of  an  excess  of 
alkali.^  After  the  internal  administration  of  copaiba,  the  urine 
turns  pink  or  rose-coloured  on  the  addition  of  hydrochloric  acid 
and  shows  three  absorption  bands,  one  (narrow)  in  the  orange  to 
the  red  side  of  D,  one  broad  band  in  the  green  between  D  and  E, 
similar  to  that  of  fuchsin,  and  one  in  the  blue.^  Tannin  leads  to 
the  appearance  in  urine  of  gallic  acid  [CeHj.  (0H)3 .  COOH],  which 
is  hence  sometimes  found  normally  in  the  urine  of  herbivora 
(horse).^  In  such  cases  the  urine  if  made  alkaline  with  caustic 
potash  turns  brown,  and  bluish-black  on  the  addition  of  ferric 
chloride.  It  also  yields  a  pink  colouration  with  Millon's  reagent, 
similar  to  that  given  by  proteids  or  tyrosin.  After  doses  of  anti- 
pyrin  [09116^20  (0113)2]  the  urine  may  be  dark-coloured  and  gives 
a  brownish-red  colour  on  the  addition  of  ferric  chloride.^  Fuchsin 
(hydrochloride  of  rosaniline  C20H19N3 .  HCl)  reappears  partly  un- 
changed in  the  urine,  to  which  it  imparts  a  reddish  tinge.  It  is 
detected  by  making  the  urine  alkaline  with  ammonia  and  shaking 
with  an  equal  volume  of  ether :  the  latter  extracts  the  colouring 
matter  and  into  the  solution  thus  obtained  a  thread  of  white  wool 
is  dipped  and  allowed  to  dry  spontaneously.  If  fuchsin  is  present 
the  wool  is  stained  red.  Salicylic  acid  (ortho-oxybenzoic  acid, 
OH .  C6II4 .  COOH)  is  excreted  partly  in  an  unaltered  form,  partly 
as  salicyluric  acid,  OH .  C6H4 .  CONH .  CH2 .  COOH.  These  may 
be  detected  by  the  intense  violet  colour  they  yield  on  the  addition 
of  ferric  chloride.  Finally  after  the  absorption  of  carbolic  acid 
(phenol)  and  many  other  aromatic  compounds  such  as  pyrocate- 
chin,  hydrochinon,  &c.,  the  urine  turns  greenish-brown  and  finally 
dark-brown  on  exposure  to  air. 


EETINAL   PIGMENTS.^ 

The  pigments  which  have  to  be  considered  under  this  heading 
are  numerous.  There  is  in  the  first  place  the  extremely  stable 
dark -brown  colouring-matter  of  the  retinal  epithelium,  belonging 
to  that  general  class  of  pigments  known  as  melanins  (see  p.  256) 
and  called  in  this  case  fuscin.  In  addition  to  this  the  retinal 
epithelium  of  some  animals  contains  a  not  inconsiderable  amount 
of  fat  globules  whose  yellow  colour  is  due  to  lipoclirin,  a  pigment 

1  For  discrimination  of  these  see  Munk,  Virchow's  Arch.  Bd.  lxxii.  (1878), 
S.  136. 

2  Quincke,  Arch.  f.  exp.  Path.  u.  Pharm.  Bd.  xvii.  (1883),  S.  273. 

3  Baumann,  Zt.  f.  physiol.   Chem.  Bd.  vr.  (1882),  S.  193. 

*  Umbach,  Arch.f.  exp.  Path.  u.  Pharm.  Bd.  xxi.  (1886),  S.  161. 

^  The  following  account  of  these  pigments  is  based  upon  Kiihne's  article  in 
Hermann's  Hdbch.  d,  Physiol.  Bd.  in.  Thl.  1.  1879,  and  on  the  original  papers  in 
Kiihne's  Untersuch.  a.  d.  physiol.  hist,  zu  Heidelberg,  1878 — 1882,  in  which  the 
literature  is  fully  quoted. 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.        261 

closely  allied  to  that  of  other  fats  of  the  body  and  known  under 
the  generic  name  of  lipochromes  or  luteins.  Passing  from  the 
epithelium  to  the  retina  proper  we  find  in  the  outer  end  of  the 
inner  limb  of  the  cones  highly  coloured  fat  globules  from  which 
three  distinct  pigments  known  as  chromophanes,  also  belonging 
to  the  general  class  of  lipochromes,  may  be  obtained ;  to  these  the 
names  rliodopliane,  chlorophane,  and  xantho2Dhane  have  been  given 
in  correspondence  with  their  respective  red,  green,  and  yellow 
colours.  In  addition  to  the  above  the  outer  limbs  of  the  rods  (not 
the  inner  limbs  or  either  the  inner  or  outer  limbs  of  the  cones) 
after  the  retina  has  been  shielded  for  some  time  from  the  action 
of  light,  are  found  to  present  a  distinct  reddish-purple  colour 
which  is  very  marked  when  the  retina  is  examined  as  a  whole. 
This  colour  ^  is  due  to  an  exceedingly  unstable  ^  pigment  called  by 
Klihne  '  visual-purple '  or  rhodopsin.  The  stability  of  the  above 
pigments  other  than  visual-purple  is  merely  relative  not  absolute, 
since  they  are  all  sooner  or  later  destroyed  (bleached)  by  suffi- 
ciently prolonged  exposure  to  light.  The  possibilities  hereby 
suggested  of  a  photochemical  explanation  of  retinal  excitation 
have  however  as  yet  thrown  no  real  light  on  the  nature  of  the 
process.  It  may  be  that  the  impulses  result  from  the  changes 
which  these  pigments  undergo,  and  it  is  possible  that  the  coloured 
globules  of  the  cones  play  a  part  in  the  whole  process  not  merely 
by  the  instability  of  their  colours  but  also  by  acting  as  coloured 
though  transparent  screens,  and  thus  at  the  same  time  determin- 
ing the  advent  to  the  photochemical  apparatus  of  rays  of  certain 
wave-length  only.  Such  speculations  are  interesting  but  for  the 
present  devoid  of  any  decisive  experimental  support  (§  773). 

1.     Fuscin  (Eetinal  melanin). ^ 

This  pigment  is  found  as  minute  granules  imbedded  in  the  cell- 
substance  and  processes  of  the  retinal  epithelium  (see  §  746). 
These  granules  may  be  either  irregular,  as  they  always  are  in  the 
choroid,  or  may,  especially  as  in  birds,  possess  an  elongated  form 
with  sharply  pointed  ends  distinctly  suggestive  of  a  crystalline 
structure.  It  is  obtained  by  extracting  the  tissues  with  boiling 
alcohol,  ether,  and  water,  and  then  digesting  for  some  time  with 
trypsin.  The  residue  is  freed  from  nucleins  by  dissolving  the 
latter  in  caustic  alkalis,  and  from  neurokeratin  (p.  87)  by  decanta- 
tion  and  straining  through  fine  gauze.  The  pigment  when  freshly 
prepared  is  practically  insoluble  in  all  ordinary  reagents,  but  is 
partially  dissolved  if  boiled  for  some  time  with  strong  caustic 
alkalis  or  sulphuric  acid.     By  prolonged  treatment  with  dilute 

1  Eirst  observed  in  the  retina  of  vertebrates  by  H.  Miiller  (1851),  and  extended 
by  Leydig  in  1857. 

2  The  instability  on  exposure  to  light  was  first  described  by  Boll,  1876. 

^  The  pigments  of  the  retinal  epithelium  and  choroid  are  apparently  identical. 


262  LIPOCHRIN. 

nitric  acid  it  becomes  soluble  in  alkalis,  yielding  yellow  solutions. 
It  becomes  similarly  soluble  by  prolonged  exposure  to  light  with 
free  access  of  air  (oxygen)  and  may  be  again  precipitated  from 
these  solutions  by  the  addition  of  an  acid.  It  is  remarkable  that 
notwithstanding  its  extreme  insolubility  and  resistance  to  the 
action  of  most  reagents  fuscin  is  gradually  bleached  by  exposure 
to  light,  a  result  due  to  some  oxidational  change  since  it  only 
occurs  in  presence  of  oxygen.  The  product  to  which  the  above 
description  refers  contains  much  nitrogen,  and  leaves  on  incinera- 
tion a  slight  ash-residue  containing  traces  of  iron. 

Later  investigations  of  the  pigment  (from  the  choroid  and  iris)  con- 
firm the  above  statements  of  its  insolubility  in  most  reagents,  and 
further  show  that  it  contains  neither  sulphur  nor  iron.  The  black 
pigment  from  hairs  is  stated  to  contain  less  nitrogen  and  a  not  incon- 
siderable amount  of  sulphur  but  no  iron,  and  to  be  readily  soluble  in 
alkalis.^  When  the  several  substances  described  under  the  general 
term  melanins  are  compared  each  with  the  other  it  is  found  that  they 
are  by  no  means  identical,  but  in  the  absence  of  any  guarantee  of  the 
purity  of  each  product  or  of  the  absence  of  change  during  its  prepara- 
tion, all  sj)ecific  statements  of  differences  must  be  received  with  caution. 
Possibly  they  are  all  closely  allied  and  probably  in  some  cases,  as  in 
the  melanjemia  of  the  malarial  fever  ^  or  the  melanuria  (and  melanotic 
pigmentation)  accompanying  certain  kinds  of  tumours  (p.  256),  they 
are  derived  from  the  colouring-matter  of  the  blood.  The  divergence 
in  views  as  to  their  derivation  from  hfemoglobin  has  apparently  turned 
in  many  cases  on  the  presence  or  absence  of  iron  in  the  pigments  un- 
der examination.  Some  of  the  melanins  may  contain  iron,  some  none, 
but  whether  they  do  or  do  not  is  not  a  decisive  test  of  their  derivation. 
If  they  do  it  makes  the  connection  more  probable,  if  they  do  not  they 
may  still  take  their  origin  from  blood-pigments,  as  in  the  case  of  the 
highly  coloured  but  iron-free  hsematoporphyrin. 

2.     Lipochrin. 

The  fat  globules  in  the  retinal  epithelium  from  which  this  pig- 
ment is  obtained  are  more  especially  abundant  in  the  frog.  It  is 
soluble  in  chloroform,  ether,  benzol,  carbon  bisulphide,  &c.  When 
dissolved  in  ether  it  gives  two  absorption  bands  between  F  and  G ; 
in  carbon  bisulphide  two  bands,  one  each  side  of  F.^  The  pigment 
of  the  body-fat  of  frogs  gives  similar  absorption  spectra  when 
dissolved  in  the  same  solvents.  Solutions  of  lipochrin  are  slowly 
bleached  by  exposure  to  a  strong  light.  The  pigment  is  probably 
closely  allied  to  the  yellow  colouring-matter  of  many  other  animal 
fats.     (See  below  sublutein.) 

1  Sieher,  Arch.f.  exp.  Path.  u.  Pharm.  Bd.  xx.  (1886),  S.  362. 

2  For  references  see  Gamgee,  Phi/sioL  Chem.  Vol.  i.  (1880),  p.  162. 

3  See  Kuhne  and  Ayres,  Jl.  of  Physiol.  Vol.  i.  (1878),  p.  109. 


CHEMICAL  BASIS  OF  THE  ANIMAL  BODY.  263 

3     Chromophanes.^ 

These  are,  as  stated  above,  the  colouring-substances  of  the  fat- 
globules  which  occur  between  the  outer  and  inner  limbs  of  the 
retinal  cones.  They  are  prepared,  as  yet  chiefly  from  the  eyes  of 
birds,  as  follows.  The  retinas  are  dehydrated  with  alcohol  and 
extracted  with  ether.  The  ethereal  solution  of  the  fats  is  then 
evaporated  to  dryness,  the  residue  dissolved  in  hot  alcohol  and 
saponified  with  caustic  soda.  The  hard  coloured  soaps  thus 
obtained  are  then  extracted  in  succession  with  petroleum  ether 
(see  note  p.  156),  ether,  and  benzol ;  of  these  solvents  the  first 
dissolves  out  the  yellowish-green  chlorophane,  the  second  the 
yellow  xanthophane,  and  the  third  the  red-coloured  rhodophane. 

(i)  CliloToijhane.  Soluble  in  petroleum  ether,  ether,  carbon 
bisulphide,  and  in  alcohol.  When  dissolved  in  the  first  two  of 
these  solvents  it  shows  two  absorption  bands  between  F  and  G ; 
in  solution  in  the  latter,  the  two  bands  lie  one  each  side  of  F. 

(ii)  Xanthophane.  Soluble  in  ether,  carbon  bisulphide,  and  in 
alcohol.  In  ethereal  solution  it  shows  only  one  absorption  band, 
near  F,  towards  the  blue  end  of  the  spectrum.  In  carboi]  bi- 
sulphide it  shows  similarly  one  band  near,  and  to  the  blue  side 
of,  h.  It  is  thus  distinguished  from  the  yellow  pigment  (lipochrin) 
of  the  retinal  epithelium  previously  described. 

(iii)  Rhodoijhane.  Soluble  in  turpentine,  benzol,  and  in  alcohol. 
In  benzolic  solution  it  shows  one  band  close  to,  but  on  the  red 
side  of,  F ;  in  solution  in  turpentine  the  band  is  similarly  near, 
but  now  on  the  blue  side  of,  F. 

Solutions  of  the  chromophanes  are  slowly  bleached  by  the  ac- 
tion of  light,  —  chlorophane  losing  its  colour  fairly  rapidly,  xantho- 
phane more  slowly,  and  rhodophane  only  after  prolonged  exposure. 
In  the  less  pure  form  in  which  the  chromophanes  were  first  ob- 
tained by  Kiihne,  they  gave  the  reactions  which  characterise  the 
lipochromes  or  lutein,  viz.  :  (i)  A  transient  violet,  followed  by  a 
bright  blue,  when  treated  with  concentrated,  sulphuric  acid,  (ii)  A 
transient  bluish-green  under  the  influence  of  strong  (yellow)  nitric 
acid,  (iii)  An  initial  green  colour,  passing  into  bluish-green,  by  the 
action  of  a  dilute  (-25  p.  c.)  solution  of  iodine  in  dilute  (-5  p.  c.) 
iodide  of  potassium.^  In  the  purer  form  in  which  they  were  sub- 
sequently prepared,  Kiihne  found  that  they  all  three  gave  the 
first  of  the  above  reactions,  while  none  of  them  were  coloured  by 
the  iodine  solution,  and  in  the  case  of  rhodophane  the  second 
reaction  with  nitric  acid  was  scarcely  marked. 

1  Kiihne  and  Ayres,  loc.  cit.  and  ibid.  p.  189. 

2  See  Capranica,  Arch.  f.  Physiol.  1877,  S.  283.  For  a  conclusive  reply  to  the 
views  as  to  the  identity  of  these  fatty  pigments  with  lutein,  put  forward  in  this  paper, 
see  Kiihne,  Unters.  a.  'd.  phi/sioL  Instit.  Heidelb.  Bd.  IT.  (1882),  S.  169. 


264  VISUAL-PUEPLE. 

4.     Visual-purple  {Ehodopsin). 

This  extremely  unstable  pigment  may  be  stated  to  occur  gen- 
erally (some  few  exceptions  have  been  observed)  in  the  retin?e  of 
all  vertebrates.  It  does  not  appear  as  yet  to  have  been  found 
in  the  eye  of  invertebrates.^  It  is  confined  entirely  to  the  outer 
limbs  of  the  rods,  but  while  occurring  in  the  majority  of  the  rods 
it  is  not  found  in  all  of  them ;  thus,  it  is  absent  in  those  situated 
in  the  immediate  neighbourhood  of  the  ora  serrata,  and  (in  man 
at  least)  it  is  wanting  in  the  scantily  disposed  rods  in  the  imme- 
diate neighbourhood  of  the  fovea  centralis.  It  is  entirely  absent 
from  the  cones,  and  hence  is  not  found  either  in  the  fovea  cen- 
tralis of  the  human  retina,  or  in  the  rod-free  retina  of  reptiles. 

Preparation  in  solution.  The  most  suitable  material  is  afforded 
by  the  retinse  of  frogs  which  have  been  kept  in  the  dark  for  two 
or  three  hours ;  since  in  these  animals  not  only  is  the  visual-pur- 
ple very  marked  and  somewhat  persistent  under  the  action  of  light, 
but  further,  the  retina  can  be  separated  from  the  adjacent  epithelium 
with  great  ease  and  is  free  from  blood.  The  necessary  operation 
for  the  removal  of  the  retina^.,  as  also  all  subsequent  manipula- 
tions, must  be  carried  on  in  a  feeble  light  from  a  sodium  flame 
to  avoid  bleaching.  The  retinae  (20 — 30  suffice)  are  then  extracted 
for  an  hour  in  the  dark  with  about  1  c.c.  of  a  freshly  prepared 
2 — 5  p.  c.  solution  of  bile  salts  from  ox-bile,  which  is  finally  fil- 
tered. If  brought  into  daylight  and  examined,  the  solution  is 
seen  to  possess  a  brilliant  pinkish-purple  colour,  which  rapidly 
becomes  red,  yellow,  and  finally  colourless,  under  the  action  of 
light.  A  similar  initial  colour  is  observed  in  the  retina  in  situ, 
followed  by  the  same  change  of  colour  when  exposed  to  light, 
the  yellow  being  regarded  as  due  to  a  '  visual-yellow '  (xanthopsin) 
and  perhaps  the  final  colourless  stage,  since  it  admits  of  regenera- 
tion in  the  dark  into  visual-purple  if  the  retina  is  fresh  and  in 
contact  with  its  epithelium  (see  §  773),  may  be  spoken  of  as  a 
'  visual-white '  (leukopsin). 

Spectroscopic  properties.  Neither  visual-purple  nor  visual-yel- 
low gives  any  distinct  absorption  band ;  there  is  a  general  absorp- 
tion of  the  central  parts  of  the  spectrum  easily  seen  between  J^  and 
G  in  the  case  of  visual-purple,  which  changes  into  a  general  absorp- 
tion of  the  violet  end  of  the  spectrum  from  F  onwards  as  the 
purple  changes  into  yellow  and  finally  disappears  altogether. 

Action  of  light.  White  light,  as  also  that  from  an  electric 
lamp  or  magnesium  flame,  bleaches  visual-purple  with  extreme 
rapidity,  dependently  upon  the  intensity  of  the  illumination: 
direct    sunlight    destroys    the    colour    almost    instantaneously. 

^  The  red  colour  of  the  retina  of  Cephalopods,  first  described  by  Krohn  iu  1 839, 
is  due  to  other  pigments  which  are  very  resistant  to  the  action  of  light. 


CHEMICAL  BASIS   OF  THE   ANIMAL   BODY.        265 

When  monochromatic  light  (of  the  spectrum)  is  used,  it  is 
found  that  the  yellowish-green,  i.  e.  the  region  most  strongly 
absorbed  by  the  pigment,  is  most  active,  followed  seriatim  by 
green,  blue,  greenish-yellow,  yellow,  violet,  orange,  and  red :  the 
ultra-red  rays  have  no  such  bleaching  power.  At  low  tem- 
peratures the  effect  of  light  is  less,  increases  with  rise  of  temper- 
ature, and  at  75°  the  colour  is  destroyed  even  without  exposure 
to  light. 

Action  of  reagents.  The  colour  is  at  once  destroyed  by  the 
action  of  caustic  alkalis,  most  acids,  alcohols,  chloroform,  and 
ether :  it  is  on  the  other  hand  persistent  in  presence  of  ammonia, 
solutions  of  ordinary  alum,  of  sodium  chloride,  carbonates  of  the 
alkalis,  and  a  large  number  of  other  salts.  One  of  the  most  im- 
portant factors  in  determining  the  bleaching  of  visual-purple  by 
either  light  or  heat  is  the  presence  or  absence  of  water.  If  the 
entire  retina  be  dried  in  vacuo  over  sulphuric  acid,  or  if  a  solution 
of  the  pigment  be  similarly  evaporated  to  dryness,  the  visual 
purple  is  comparatively  resistent  to  the  action  of  light,  although 
it  is  bleached  by  a  sufficiently  prolonged  exposure. 


LIPOCHKOMES   OE   LUTEINS. 

After  the  rupture  of  the  ovarian  follicle  which  accompanies  the 
discharge  of  an  ovum,  the  cavity  of  the  follicle  becomes  filled 
with  a  mass  of  cells,  traversed  by  ingrowths  of  connective  tissue 
from  the  neighboring  stroma,  and  frequently  contains  blood 
resulting  from  hsemorrhage  at  the  time  of  rupture  (§  934).  This 
is  followed,  most  strikingly  if  impregnation  of  the  discharged 
ovum  takes  place,  by  a  fatty  degeneration  of  the  contained  cells, 
resulting  in  the  formation  of  a  bright  pigmented  mass  of  a  bril- 
liant yellow  or  orange  colour,  while  at  the  same  time  the  colour- 
ing-matter of  the  blood  may  be  converted  into  that  crystalline 
substance  already  described  under  the  name  hsematoidin  (p.  239) 
as  being  identical  with  bilirubin.  The  structure  which  results 
from  the  above  changes  is  known  as  a  '  corpus  luteum.'  The  earlier 
(1868)  examination  of  coloured  extracts  of  these  corpora  lutea 
led  to  erroneous  statements  of  the  identity  of  the  pigment  ob- 
tained from  them  with  hsematoidin,  —  a  view  which  was  almost 
immediately  contested,  —  while  the  colouring  matter  received  the 
name  of  hsemolutein.  A  renewed  investigation  of  the  pigment  led 
Thudichum  ^  to  characterise  it  as  of  wide-spread  occurrence  in  the 
highly  coloured  fatty  constituents  as  of  butter,  fats,  egg-yolk,  &c., 
and  of  some  vegetable  tissues,  and  to  give  it  the  name  lutein,  under 

1  Centralb.f.  d.  med.  Wiss.  1869,  S.  1. 


266  LUTEINS. 

which  designation  as  a  class-name  these  fatty  pigments  have 
usually  been  known.  Since,  however,  as  we  have  already  seen 
in  the  case  of  the  chromophanes,  and  as  will  appear  subsequently 
in  the  case  of  the  pigments  of  egg-yolk,  and  of  the  substance 
tetronerythrin,  we  have  to  deal  with  pigments  which,  while  they 
give  the  reactions  characteristic  of  the  group,  exhibit  colours 
other  than  yellow,  it  is  perhaps  advisable  now  to  use  the  term 
'  lipochrome '  as  generic,  and  to  retain  lutein  as  specific  for  certain 
yellow  pigments  only.  The  lipochromes  are  characterised  by 
exhibiting  absorption  bands  which,  though  varying  somewhat  in 
position  according  to  the  solvent  employed,  are  usually  situated 
towards  the  violet  end  of  the  spectrum.  From  a  chemical  point 
of  view  the  reactions  already  described  on  p.  263  may  be  regarded 
as  characteristic  of  the  whole  class. 

1.  Lutein.^ 

This  pigment  may  be  obtained  from  corpora  lutea  by  extraction 
with  chloroform.  If  the  orange-coloured  solution  thus  obtained 
be  allowed  to  evaporate  spontaneously,  a  fatty  residue  is  left  in 
which  the  lutein  is  found  in  a  crystalline  form,  as  minute  either 
rhombic  prisms  or  plates,  which  are  pleochromatic  (see  p.  216). 
They  are  insoluble  in  water,  but  readily  soluble  in  alcohol,  ether, 
chloroform,  and  benzol.  These  exhibit  two  absorption  bands,  one 
inclosing  F,  the  other  about  half  way  between  F  and  G. 

If  egg-yolk  be  extracted  with  a  little  alcohol  and  much  ether, 
the  solution  shows  two  bands  similar  to  those  already  described 
for  lipochrin  or  frog's  fat  (p.  262),  while  sometimes  a  third  faint 
band  near  G  may  be  seen,  especially  if  the  residue  from  the 
ethereal  extract  be  dissolved  in  carbon  bisulphide  and  examined. 
If  the  residues  from  the  ethereal  extracts  of  egg-yolk  and  corpora 
lutea  be  saponified  and  extracted  with  carbon  bisulphide,  the 
solutions  yield  identical  absorption  spectra.^ 

Maly,^  operating  on  the  bright  red  eggs  of  a  sea-spider  (Maja 
Squinado)  considered  that  lutein  (assuming  its  identity  in  this 
case  with  that  from  ordinary  egg-yolk)  consists  of  two  pigments, 
vitellolutein  (yellow)  and  vitellorubin  (red).  For  further  details 
see  the  original  paper.  Lutein  is  more  or  less  rapidly  bleached 
by  the  action  of  light. 

2.  Serum  lutein. 

The  serum  from  the  blood  of  almost  all  animals  is  usually  of 
a  more  or  less  yellow  colour ;  it  is  specially  marked  in  the  case 
of  the  horse  and  ox,  is  also  marked  in  the  case  of  sheep  and  man, 
and  is  but  slightly  present  under  normal  conditions  in  the  serum 

1  See  Capranica,  loc.  cit.  on  p.  263. 

2  Kiihne  and  Ayres,  Jl.  of  Physiol.  Vol.  i.  (1878),  p.  127.     Gives  spectra. 

3  Monatshefte  f.  Chem.  Bd.  ii.  (1881),  S.  18.  Gives  literature  to  date.  See 
recently  Bein*  Ber.  d.  d.  chem.  Gesell.  Bd.  xxiii.  (1890),  S.  421, 


CHEMICAL  BASIS   OF   THE   ANIMAL  BODY.        267 

of  the  dog,  rabbit,  or  cat.  The  colour  has  by  different  observers 
been  ascribed  to  different  pigments.  In  some  cases  it  may  be 
due,  at  least  partly,  to  the  presence  of  bile-pigments  or  their 
derivatives,^  these  being  much  increased  in  certain  diseases,  such 
as  jaundice.  But  in  addition  to  these  it  appears  that  the  colour 
of  all  pigmented  serums  is  due  to  a  specific  pigment,  which,  while 
it  may  differ  (?)  slightly  as  obtained  from  the  blood  of  different 
animals,  belongs  in  each  case  to  the  general  class  of  substances 
known  as  lipochromes.  This  view  was  originally  put  forward  by 
Thudichum,^  who  ascribed  the  colour  to  the  pigment  lutein,  which 
has  been  already  described.  This  view  is  probably  correct,  inde- 
pendently of  the  possibility  that  the  colour  may  be  in  some  cases 
due  partly  to  the  simultaneous  presence  of  bile-pigments  or  their 
derivatives.  Thus  it  is  found  ^  that  by  shaking  serum  with  ethyl 
or  amyl  alcohol  a  coloured  extract  is  obtained  which  contains  a 
fatty  pigment,  evidently  belonging  to  the  class  of  lipochromes,  as 
judged  by  the  fact  that  it  is  soluble  in  alcohol,  ether,  chloro- 
form, benzol,  carbon  bisulphide,  &c.,  shows  the  two  (in  the  case  of 
birds  only  one)  bands  in  the  blue  part  of  the  spectrum,  and  giyes 
the  chemical  reactions  (p.  263)  with  nitric  acid  and  sulphuric  acid 
characteristic  of  these  substances.  It  is  in  many  cases  identical 
with  the  pigment  which  can  be  extracted  from  the  fat  of  the 
animal  from  whose  blood  the  serum  was  obtained.  Serum-lutein 
is  bleached  by  the  action  of  light. 

3.     Tetronerythrin. 

This  name  was  first  given  to  a  substance  extracted  by  chloro- 
form from  the  red  excrescences  over  the  eyes  of  certain  birds.* 
It  was  subsequently  investigated  by  Hoppe-Seyler  (from  the 
same  source),  and  described  later  as  occurring  in  some  sponges,^ 
fishes,^  and  feathers.'''  More  recently  it  has  been  found  as  a  pig- 
mentary constituent  of  the  blood  of  Crustacea.^  The  pigment  is 
readily  soluble  in  alcohol,  ether,  chloroform,  benzol,  and  carbon 
bisulphide,  is  readily  bleached  by  light,  yields  the  chemical  reac- 
tions with  sulphuric  acid,  nitric  acid,  and  iodine,  which  are  char- 
acteristic of  the  lipochromes  (see  p.  265),  like  these  shows  an 
absorption  band  near  F  somewhat  similar  to  that  of  xanthophane 

1  Hammarsten  (Swedish).  See  Malj-'s  Jahresb.  1878,  S.  129  (Bilirubin  in  blood- 
serum  of  horse  but  not  of  ox  or  man).  Maly,  Liebig's  Annal.  Bd.  163  (1872),  S.  77 
(Hvdrobilirubin).    MacMunn,  Proc.  Roy.  Soc.  Vol.  xxxi.  (1880),  p.  231  (Choletelin). 

2  Centralh.f.  d.  med.  Wiss.  1869,  S.  1. 

3  Krukenberg,  Sitzb.  d.  Jena.  Gesell.  f.  Med.  u.  Naturwiss.  1885.  Halliburton, 
Jl.  of  Physiol.  Vol.  vii.  (1885).  p.  324. 

*'  Wurm,  Zt.  f.  wiss.  Zool.  Bd.  xxxi.  (1871),  S.  535. 

5  Krukenberg,  Centralb.  f.  d.  med.  Wiss.  1879,  S.  705. 

6  Krukenberg,  Vergleicli.-physiol.  Stud.  1  Reihe,  Abth.  4,  1881,  S.  30. 

7  Krukenberg,  Ibid.  Abth.  5,  S.  87.  2  Reihe,  Abth.  1,  S.  151.  See  also  Merej- 
kowski,  Compt.  Rend.T.  xciii.  (1881),  p.  1029.  Mac  Munn,  Proc.  Roy.  Soc.  Vol. 
XXXV.  (1883),  pp.  132,  370. 

8  Halliburton,  Jl.  of  Physiol.  Vol.  vi.  (1884),  p.  324. 


268  PYOCYANIN. 

and  rhodophane  (p.  263),  and  is  slowly  bleached  by  the  action  of 
light. 

The  pigments  of  the  animal  body  which  have  been  so  far  dealt 
with  admit  of  a  certain  amount  of  classification  with  reference 
either  to  the  secretions  or  organs  in  which  they  occur,  to  their 
genetic  relationships  each  with  the  other,  or  in  some  cases  (lipo- 
chromes)  to  their  probable  chemical  similarities.  But  in  addition 
to  these  an  extremely  numerous  mass  of  pigments  has  been  at  dif- 
ferent times  described  under  various  names,  as  obtained  from  the 
brightly-coloured  parts  of  invertebrates  and  of  vertebrates,  such  as 
the  feathers,  &c.  Our  knowledge  of  them  is  quite  incomplete  and 
limited  in  most  cases  to  statements  of  their  solubilities  and  the 
absorption  spectra  which  some  of  them  yield.  In  most  cases  nothing 
is  known  of  their  chemical  nature  or  their  relationships  (if  any) 
to  each  other,  and  any  description  of  them  even  if  it  were  profit- 
able, is  impossible  within  any  reasonable  limits. 

For  details  and  references  to  the  literature  of  the  several  pigments 
see  Gamgee,  Physiological  Chemistry,  Vol.  i.  1880,  p.  305,  and  parti- 
cularly Krukenberg,  Vergleichend-physiol.  Studien,  Heidelberg,  1881- 
1888  and  Vergleich.  physiol.   Vortrdge,  Bd.  i.  1886,  Nr.  3. 

In  conclusion  it  must  suffice  to  describe  two  pigments  which  do 
not  naturally  fall  under  any  of  the  above  groups  into  which  these 
substances  have  been  divided. 

Pyocyanin}  Pus,  which  ordinarily  presents  a  more  or  less 
bright  yellow  colour,  is  frequently  greenish  and  sometimes  blue. 
The  blue  colour  is  due  to  a  pigment  (pyocyanin)  which  is  ap- 
parently formed  in  the  pus  by  the  action  of  specific  organisms. 
It  is  obtained  either  from  pus  or  the  bandages  into  which  it  has 
been  absorbed  by  extraction  with  dilute  alcohol  or  with  water  to 
which  a  trace  of  ammonia  has  been  added.  The  alcoholic  extract 
is  then  evaporated  to  a  small  bulk  and  the  residue  extracted  with 
chloroform,  or  it  may  be  extracted  at  once  from  the  aqueous  solu- 
tion by  shaking  with  chloroform.  It  may  be  obtained  in  a  crys- 
talline form  by  slow  evaporation  of  the  chlorof ormic  solutions,  the 
crystals  being  readily  soluble  in  water  and  alcohol,  but  only  slightly 
in  ether.  Acids  change  the  blue  colour  to  red,  and  alkalis  restore 
the  original  blue.  None  of  the  solutions  show  any  distinct  ab- 
sorption bands.  When  kept  the  crystals  turn  greenish,  due  to  a 
decomposition  which  takes  place  most  readily  in  alkaline  solu- 
tions exposed  to  the  air  and  light,  and  results  in  the  formation  of 
a  yellow  pigment,  pyoxanthose.     The  latter  is,  unlike  pyocyanin, 

1  Fordos,  Compt.  Rend.  T.  li.  (1860),  p.  215;  Ibid.  lvi.  (1863),  p.  1128.  Liicke, 
Arch.  f.  klin.  Chirurg.  Bd.  iii.  (1863),  S.  135.  Girard,  Deutsch.  Zeit.  f.  Chirurg. 
Bd.  VII.  (1876),  S.  389.  Fitz,  Ber.  d.  d.  chem.  Gesell.  Bd.  xi.  (1878),  Sn.  54,  1893. 
Kunz.  Monatsh.f.  Chem.  Bd.  ix.  (1888),  S.  361. 


CHEMICAL  BASIS   OF   THE  ANIMAL  BODY.        269 

only  slightly  soluble  in  water,  but  readily  soluble  in  etlier,  by 
which  property  the  two  pigments  admit  of  being  separated. 
Pyoxanthose  is  crystalline,  soluble  in  alcohol  and  chloroform,  is 
coloured  red  by  acids  and  violet  by  alkalis.  Since  pyoxanthose 
appears  to  be  a  product  of  the  decomposition  of  pyocyanin,  both 
pigments  may  occur  simultaneously  in  pus,  in  which  case  the 
fluid  is  green.  According  to  some  more  recent  observations  ^  pyo- 
cyanin, as  judged  of  by  its  reactions  with  the  chlorides  of  gold  and 
platinum  and  with  other  alkaloidal  precipitants,  as  also  from  the 
formation  of  crystalline  compounds  with  acids,  is  closely  related  to 
the  alkaloids. 

Sweat  is  also  occasionally  coloured  blue,  in  some  cases  by  in- 
digo-blue (p.  200)  as  in  urine,  and  it  may  be  (?)  by  a  pigment 
similar  to  pyocyanin. 

Pigment  of  the  suprarenal  bodies.  A  suprarenal  body  when  a 
section  is  made  through  it  is  found  to  consist  of  an  outer  or  corti- 
cal portion,  of  a  yellow  colour,  which  constitutes  the  chief  part  of 
its  structure,  and  an  inner,  medullary  part  of  a  darker  colour. 
When  the  latter  is  acted  upon  by  ferric  chloride  it  assumes  a 
dark  bluish-  or  greenish -black  colour,  and  if  an  aqueous  extract 
of  its  substance  (or  the  tissue  itself)  be  treated  with  an  oxidising 
agent  it  turns  red  (§498).  It  appears  therefore  that  the  supra- 
renals  contain  some  form  of  chromogen  or  pigment-forerunner 
which  gives  rise  under  appropriate  conditions  to  a  pigment.  Ac- 
cording to  some  observers  extracts  of  the  cortex  show  a  spectrum 
similar  to  that  of  the  histohsematius  (p.  234)  while  the  medulla 
gives  one  resembling  hsemochromogen.^  The  pigment  obtainable 
from  the  suprarenals  has  been  investigated  by  Krukenberg.^  By 
a  method  for  which  the  original  paper  must  be  consulted,  he  iso- 
lated a  brownish-red  substance  with  an  acid  reaction,  soluble  in 
water  and  alcohol,  whose  reactions  were  the  same  as  those  of  ex- 
tracts of  the  suprarenals.  None  of  the  solutions  showed  any  dis- 
tinct absorption  bands.  The  whole  subject  requires  further 
investigation,  which  might  be  of  interest  in  connection  with  the 
origin  and  causation  of  the  increaesd  pigmentation  of  the  skin  ob- 
served when  the  suprarenals  are  diseased. 

1  Gessard,  Compt.  Rend.  T.  xciv.  (1882),  p.  536. 

2  MacMunn,  Proc.  Physiol.  Soc.  Dec.  1884  (Jl.  of  Physiol.  Vol.  v.  p.  xxiv). 

3  Virchow's  Arch.  Bd.  ci.  (1885),  S.  542. 


INDEX. 


'  Absorption  ratio, '  225,  note 

Acetamide,  formatiou  of,  160 

Acetic  acid,  116 

Acetone,  117 

Achroodextrin,  preparation  of,  93 

Acid,  «-amido-caproic,  147 

,,  acetic,  116,  124 

,,  allanturic,  170 

,,  amido-acetic,  140 

,,  amido-caproic,  147 

„  amido-ethylsulphonic,  141 

,,  amido-formic,  139 

,,  aniido-pyro-tartaric,  152 

,,  amido-succinamic,  153 

,,  amido-succinic,  152 

,,  amido-sulpholactic  (cystin),  150 

,,  amido-valerianic,  85 

,,  aspartic  or  asparaginic,  152 

,,  benzoic,  186 

,,  butyric,  118,  124 

,,  capric,  118 

5,  caproic,  118 

,,  caprylic,  11^ 

,,  carbamic,  151 

,,  carbolic  or  })henylic,  193 

„  cholalic  or  cholic,  207 

,,  choleic,  209 

,,  ethylene-lactic,  129 

,,  ethylidene-lactic,  125 

,,  fellic,  209 

„  formic,  116,  124 

,,  glutamic,  152 

,,  glycerinpliosphoric,  135 

»  glycocliolic,  210 

„  glycolic,  124 

„  glycuronic,  107 

,,  hippuric,  186 

,,  hydantoic,  170 

,,  hydrochloric,  percentage  of  in  gas- 
tric juice,  61 

,,  hydroxy-butyric,  130 

,,  hydroxy-propionic,  124 

,,  indoxyl-sulphuric,  199 

,,  isethionic,  141 

,,  isobutyric,  118 

,,  kresylsulphuric,  195 

,,  kynurenic,  192 


Acid,  lactic,  124 

,,     lauric  or  laurostearic,  119 

„     'lithic,'166 

,,     margaric,  119 

,,     methyl-guanidinacetic,  143 

,,     methyl-hydantoic,  141 

,,     myristic,  119 

,,     oleic,  120 

,,     oxalic,  130 

,,     oxaluric,  171 

,,     oxychinolin-carboxylic,  192 

,,     palmitic,  119 

,,     parabanic,  169 

,,     paralactic,  126 

,,     phenylic,  193 

,,     phenyl-sulphuric,  194 

,,     propionic,  117,  124 

,,     saccharic,  107 

,,     sarcolactic,  126,  128 

,,     skatoxyl-sulphuric,  202 

,,     stearic,  119 

,,     succinic,  131 

,,     sulpho-cyanic,  163 

,,     sulphuric,  53 

,,     tauro-carbamic,  143 

,,     taurocholic,  211 

5,     uric,  164 

,,     valeric  or  valerianic,  118 
Acid-Albimiin,  15 

,,     its    relation   to    alkali-albumin, 
18 
Acids  of  the  acetic  series,  115 

,,      aromatic  series,  185 
,,      glycolic  series,  124 
,,      oleic  (acrylic)  series,  120 
,,      oxalic  series,  130 
fatty,  115 
Acrolein,  121 

Acrylic  series,  acids  of  the,  120 
Adamkiewicz's  reaction  for  proteids,  8 
•Adenin,'  89,  174,  181 
Adipocire,  formation  of,  119 
iEthalium  septicum,  4 

,,  ,,     glycogen    present    in, 

95-96 
Albumin,  its  decomposition  by  acids  and 

enzymes,  40 


272 


INDEX. 


Albumins,  derived  and  native,  9 
,,  chemistry  of,  11,  15 

,,         preparation  of,  12,  16 
Albuminates,  15 
'  Albuminose,'  37 
Albumoses  and  peptones,  10 

,,  ,,       cheraistry  of,  36 

,,  ,,       preparation  of,  42 

Alcohols  of  the  human  body,  116 
Aldehydes,    their    possible    presence   in 
plants,  52,  115,  note 
,,      their  relations  to  the  ketones,  117 
Aleurone-grains  of  plants,  26,  36 
Alkali-albumin,  9,  18 

,,  chemistry  of,  18 

,,  preparation  of,  19 

,,  rotatory  power  of,  19 

,,  its  relations  to  casein,  22 

Alkaloids,  certain  vegetable,  their  rela- 
tion to  the  xanthins,  173, 
174 
vegetable,  their  resemblance 
to  ptomaines,  204,  205 
Allantoin  series,  170 

,,       sources  of,  172 
,,       preparation,  173 
Allanturic  acid,  170 
Alloxan  series,  169 
Amides  and  amido-acids,  139 
Amido-acids  of  the  acetic  series,  139 
,5  ,,      lactic  series,  150 

,,  ,,      oxalic  series,  151 

Amido-acetic  acid,  140 
«i-amido-caproic  acid,  147 
Amido-ethylsulphonic  acid,  141 
Amido-fonnic  acid,  139 
Amido-pyro-tartaric  acid,  152 
Amido-succinamic  acid,  153 
Amido-succinic  acid,  152 
Amido-sulpholactic  acid  (cystin)  150 
Amido-valerianic  acid,  85 
Amines,  composition  of,  204,  note 
Anmionium   carbonate,    its  relations  to 

urea,  161,  163 
Amphikreatinin,  207 
Amphopeptone,  46 
Amylodextrin,  92 

Animal  body,  chemical  basis  of  the,  3 
'  Animal  gum,'  Landwehr's,  79,  84,  95 
Antialbumate,  40 

,,  characters  of,  41 

Antialbumose,  40 

,,  characters  of,  42 

Antipeptone,  39  and  note,  40 

,,  preparation  of,  45 

Apoglutin,  82,  note 
Aromatic  series,  the,  185 
Ascidians,  tunicin  prepared  from  mantle 

of,  101 
Ash  of  egg-albumin,  5 
,,    of  proteids,  6 
,,    of  casein,  20-21 
,,    of  fibrin,  34 
Asparagin,  153 


Asparagin,  its  function  in  vegetable  me- 
tabolism, 52-53,  153,  154 
Aspartic  or  asparaginic  acid,  152 

Bananas,  presence  of  isobutyric  acid  in, 

118 
Barfoed's  reagent,   composition  of,  112, 

note 
Beans,  preparation  of  inosit  from,  108 
Benzoic  acid,  186 

,,  its    relations   to    hippuric 

acid,  186 
,,  vegetable  sources  of,  188 

Benzol-glj'cin,  186 
Bile-acids,  the,  207 

,,         variations   in,    according    to 

source,  209 
, ,         Pettenkofer's  reaction  for,  213 
Bile,  the  mucin  of,  77 
,,    and   free  fatty   acids,    emulsifying 
power  of,  123 
Bile-pigments  and  their  derivatives,  239 
,,  their    relation    to    blood- 

pigments,  248,  249 
Bilicyanin,  245 

Bilirubin,  its  identity  with  heematoidin, 
239 
,,         soui'ces  of,  240 
,,         preparation  of,  241 
Biliverdin,  243 

,,  preparation  of,  243 

Blood  and  bile,  relationship  between  col- 
ouring matters  of,  237,  247,  249 
,,    dextrose  a  constituent  of,  102 
,,    presence    of    sarcolactic     acid    in, 
126 
Blood- corpuscles,  red,  proteid  constituent 
of,  28 
,,         ,,  „  colouring  matter  of, 

215 
,,         ,,  white,  their  connection 

with  fibrin  for- 
mation, 67-69 
,,         5,  ),       glvcogen  present 

in,  96 
,,         ,,  nucleated,  nuclein  pre- 

pared from,  88 
Blood-plasma,   fibrinogen   a   constituent 
of,  29 
,,  paraglobulin   a  constitu- 

ent of,  27 
Blood-stains,  detection  of,  237 
Body,  colouring  matters  of  the,  215 
Brain-substance,    neurokeratin   obtained 
from,  87 
,,  ethyl-alcohol    obtained 

from,  116 
,,  inosit  present  in,  108 

,,  a  sugar  obtained  from, 

106-107 
,,  preparation  of  cerebrin 

from,  138 
„  protagon  obtained  from, 

137 


INDEX. 


273 


Briicke's  reagent  for  proteids,  8 
Bunsen  method,  the,  of  estimating  urea, 

158 
Bush-tea,  alkaloidal  principle  of,  185 
Butter,  fats  present  in,  122 
Butyric  acid,  118 

,,       fermentation,  105 

Cadaverin,  206 

Caffein,   its  relations  to   xanthin,    173, 
174,  184-185 
,,       an     excretionary     product     of 
plants,  185 
Calcium  lactate,  126 
,,        oxalate,  130 
,,       salts,  their  action  in  clotting  of 

casein,  22 
,,       sarcolactate,  126,  127 
Calculi,  cystic,  150 

,,       mulberry,  130 
Cane-sugar,  digestive  changes  in,  59,  110 
,,  dextrose  formed  from,  105     ^ 

'inversion'  of,  106,  110 
Cane-sugar  group,  the,  110 
Capric  (rutic)  acid,  118 
Caproic  acid,  118 
Caprylic  acid,  118 
Carbamic  acid,  151 
Carbamide,  155 
Carbohydrates,  91-115 

,,  in  what  form  assimilated, 

59,  98,  103,  111,  114-115 
Carbolic  acid,  193 
Carbon-dioxide  haemoglobin,  222 
Carbon-monoxide  haemoglobin,  221 
Carica   Papaya,   peptonizing   enzyme  in 

the  juice  of,  61 
Carnin,  preparation  of,  178 
Carnivora,  nature  of  bile-acid  of,  210 
Casein,  9 

,,      chemistry  and  preparation  of,  20 
,,      action  of  rennin  on,  22 
,,      its  relations  to  nuclein,  90 
Casein  ogen,  20 
Caseoses,  25 
Caterpillars,  formic  acid  in  the  secretion 

of  certain,  116 
Caviar,  presence  of  vitellin  in,  26 
Cell-globulins,  28 
Cell-protoplasm,     presence    of    nucleo- 

albumin  in,  90 
Cell-walls,  vegetable,  lignification  of,  99 
Cells,  chemical  composition  of  nuclei  of, 
88 
,,     hepatic,  their  glycogen-con verting 
action,  98 
Cellulose  of  starch  grains,  91 
,,       chemistry  of,  99 
,,       digestion  of,  99,  100 
Celtis  reticulosa,  presence  of  skatol  in, 

203 
Cerebrin,  138 
Cerebrose,  106 
Cetyl  alcohol,  116 


Charcot's  crystals,  139 

Cheese,  curd  of,  produced  only  by  rennin, 

22,  65 
Chitin,  preparation  of,  87 
Chloroform,  disciimination  between  en- 
zymes and  ferments  by  means  of,  56 
Chlorophane,  261,  263 
Chlorophyll,    starch    formed  under  the 

intiuence  of,  91 
Cholalic  or  cholic  acid,  207 

,,  ,,  ,,       preparation,  208 

Cholecyanin  or  choleverdin,  245 
Choleic  acid,  209 
Cholesterin,  131 

,,  reactions  of,  133 

Choletelin,  245 
Cholin,  135 
Cholo-hajmatin,  250 
Chondrigen,  83 
Chondrin,  preparation  and  reactions  of, 

83 
Chondromucoid,  85 
Chromogens,  251,  256-257 
Chromophanes  of  the  retina,  261,  263 

, ,  action  of  light  on  the,  26b 

Chrysokreatinin,  207 
Chyle,  presence  of  globulins  in,  28,  29 

,,       dextrose  a  constituent  of,  102 
Clotting  of  casein,  22 

of  blood,  29,  67,  69 
,,        of  muscle  plasma,  30,  70 
,,        of  milk,  heat  phenomena  of,  75 
Collagen,  80 

,,        its  conversion  into  gelatin,  80 
Copper,  its  presence  in  animal  pigments, 

230 
Corpus  luteum,  pigment  of  the,  265 
Corpuscles,  see  Blood-corpuscles 
Crystallin,  chemistry  and  preparation  of, 

25 
Crystals,  Charcot's,  139 

,,         proteid-containing,  6 
,,         Teichmann's,  235 
Cyanogen     compounds,    their    possible 

function  in  metabolism,  52,  162 
Cystin,  150 

Deuteroalbumose,  44 
Deuterogelatose,  82 
Dextrins,  the,  preparation  of,  93 
Dextrose  (glucose,  grape-sugar),  102-106 

,,         fermentations  of,  105 

,,         discrimination  of  from  maltose, 
111 
Diabetes,  chemical  changes  in,  117,  130 
Diastase,  formation  of  maltose  by.  111 
Digestion  of  proteids,  products  of,  36 
intestinal,  58-59,  63,  64 

,,         gastric,  60 

,,         tryptic,  62-64 

„         of  cellulose,  99,  100 
Diseases,  ptomaine-formation  by  organ- 
isms characteristic  of  specific,  205 
Dysalbumose,  44 


18 


274 


INDEX. 


Dyslysin,  210 

Dyspeptone,  Meissner's,  37,  42 

Egg-albumin,  chemistry  of,  11,  89 
,,  preparation,  12 

,,  crystalline  form  of,  12 

Egg-yolk,  the  proteid  constituents  of,  26 
„         nuclein  of,  88,  90 

pigment  of,  265,  266 
Elastin,  preparation  of,  85 
Elastoses,  86 
Embryo,  presence  of  glycogen  in  tissues 

of,  96 
Enzymes,  53-76 

,,        characteristics  of,  53-56 
,,        discrimination  of  from  organ- 
ized ferments,  56 
,,        of  the  pancreas,  57 
,,         of  gastric  juice,  59 
,,        of  muscle  tissue,  70 
,,        mode  of  action,  72-74 
,,        heat   phenomena    accompany- 
ing their  action,  75 
,,        their   products    inhibitory   to 

their  action,  94 
,,        their    action     on    cane-sugar, 
110,    111 
Epidermal  structures,  keratin  the  chief 

constituent  of,  86 
Erythrodextrin,  93 
Ethane,  124 
Ethyl,  124 
Ethyl-alcohol,    presence    of,    in    animal 

tissues,  116 
Ethyl-glycol,  124 
Ethylene-lactic  acid,  129 
Ethylidene-lactic  acid,  125 
Extract  of  meat,  preparation  of  sarcolactic 
acid  from,  126-127 
,,         ,,  preparation     of     carnin 

from,    178 
,,         ,,  presence  of  hypoxanthin 

in,  179 

Fats,  their  derivatives  and  allies,  115 
,,     the  neutral,  120 
,,     complex  nitrogenous,  133 
Fattening,  sources  of  fat  deposited  dur- 
ing, 122 
Fehling's    fluid,    composition    of,    112, 

note 
Fellic  acid,  209 
Ferment,   restriction  of   the  term,    53, 

note 
Ferments,  their  probable  mode  of  action, 
72,  73 
,,         organized,   discrimination    of 
from  enzymes,  56 
Fermentations  of  dextrose,  105 

,,  lactic,   of    souring   milk, 

114 
Fibrin,  32 

,,      varying  forms  of,  33 
,,      ash  of,  33 


Fibrin,  its  action  on  hydrogen  dioxide,  35 

Fibrin-ferment,  67,  68 

Fibrinogen,  29,  30,  69 

Fibrino-plastin,  27,  note 

Fishes,  presence  of  kreatinin  in  muscles 

of,  145 
Food,  the  three  classes  of,  4 
Foot-mucin  of  Helix  pomatia,  78 
Formic  acid,  secreted  by  ants  and  certain 

caterpillars,  116 
Formica  rufa,  formic  acid  excreted  by, 

116 
Fruits,  presence  of  Isevulose  in,  106 
Fuscin,  261,  262 

Galactose,  or  cerebrose,  reactions  of,  106 

,,         a  product  of  lactose,  113 
Gall-stones,  cholesterin  a  constituent  of, 
132 
,,  bilirubin  prepared  from,  240, 

241 
Gallois'  test  for  inosit,  109 
Gastric  glands,  pepsinogen  in  the  cells 

of,  61 
Gastric  juice,  earlier  experiments  with, 
37 
,,  the  proteolytic  enzyme  of, 

59 
,,  percentage  of  hydrochloric 

acid  in,  61 
Gelatin  or  glutin,  80 

,,      liquefaction    of,   by    growth    of 
micro-organisms,  82 
Gelatin-peptones,  preparation  of,  81,  82 
Gelatoses,  the,  82 
Gland,  submaxillary,  mucin  of,  77 
Globin,  32 
Globulin  of  the  crystalline  lens,  25 

„       as  compared  with  myosin  and 
tibrin,  35 
Globulins,  the,  25-32 

,,         their     conversion    into    acid- 
albumin,  16 
,,         their  relations  to  fibrin,  34 
Glucose,  102 
Glue,  80,  note 

Glutamic  or  glutaminic  acid,  152 
Glutin,  or  gelatin,  80 
Glutoses,  the,  82 

Glycerin  (glycerol),  the  chemistry  of,  123 
Glycerinphosphoric  acid,  135 
Glycin,  glycocoll,  or  glycociue,  140 
,,       preparation  of,  140 
,,       a  product  of  gelatin  decomposi- 
tion, 81 
Glycocholic  acid,  preparation  of,  210 
Glycogen,   hepatic,    its   conversion   into 
sugar,  58,  98 
, ,  the  animal  analogue  of  starch, 

95 
,,  its  presence  in  various  tissues, 

and  in  molluscs,  95-96 
,,  preparation  of,  96 

,,  reactions  of,  97,  98 


INDEX. 


275 


Glycogen,  diminution  of,  in  muscles  dur- 
ing activity,  129 
Glycolic  acid  series,  124 
Glycosamin,  87 

Glycuronic  acid,  chemistry  of,  107 
,,  ,,     compounds  of,  107 

Gmelin's  reaction  for  bile  pigments,  242, 

245 
Gout,  accumulation  of  uric  acid  salts  in, 

164 
Grape-sugar,  chemistry  of,  102 
Guanidin  in  decomposition  of  proteids,51 
,,         its   connection   with  kreatin, 

143,  184 
,,         ,,  ,,         with  urea,  174 

,,         chemistry  of,  184 
,,         synthesis  of,  184 
Guanin,  connexions  of,  with  uric  acid, 
174 
,,      preparation  of,  182 
,,      its  conversion  into  xanthin,  183 
,,      Capranica's  reactions  for,  183 
Guano,  Peruvian,  preparation  of  uric  acid 
from,  166 
,,     preparation  of  guanin  from,  182 
Guarana,  alkaloidal  principle  of,  184-185 

Haematin,  preparation  of,  232 

,,         spectroscopy  of,  233 
Haematoidin,  239 
HiBmatoporphyrin  (iron-free  haematin), 

238 
Hasmin  (haematin-hydrochloride),  235 
Haimochromogen,  216,  231 
Haemocyaniu,  230 
Haemoglobin,  215 

,,  in   the  j)lasma  of  inverte- 

brates, 217,  note 
,,  carbon-monoxide,  221 

,,  nitric-oxide,  222 

,,  carbon-dioxide,  222 

,,  methods  of  quantitative  de- 

termination of,  224 
Helix  pomatia,  mucin  in  excretion  of,  76 

,,  the  two  mucins  of,  78 

Hemialbumose,  39,  40 

,,  characters  of,  42 

,,  preparation  of,  43 

,,  various  forms  of,  43 

Hemipeptone,  39  and  note,  40 

,,  how  obtained,  46 

Hemiprotein  of  Schiitzenberger,  39,  41 
Herbivora,  digestion  of  cellulose  by  the, 
99,  100 
„         predominance    of    stearin   in 

fat  of,  122 
,,         sources   of    hippuric   acid   in 

the,  188 
,,        pigment  of  the  bile  of  the,  242 
Heteroalbuniose,  44 
Heteroxanthin,  174,  177 
Hippuric  acid,  186 

,,         reactions,  186-187 

,,         sources  of,  in  the  herbivora,  188 


Histohaematins,  234 

Honey,  laevulose  present  in,  106 

Humus  pigments,  256 

Hydantoic  acid,  170 

Hydantoin,  170 

Hydrazones,  102 

Hydrobilirubin,  246 

, ,  its  probable  identity  with 

urobilin,  247,  252 
Hydrochinon,  197 

Hydrogen,  evolution  of,  in  butyric  fer- 
mentation, 105 
Hydroxy-butyric  acid,  130 
Hydroxy-propionic  acid,  124 
Hypoxauthin,  174 

,,  discrimination     of     from 

xanthin,  176 
,,  sources  of,  179 

,,  its    relation     to    carnin, 

178 
,,  its  relation  to    nuclein, 

180, 181 

Ichthin  and  ichthidin,  26 

Hex  Paraguayensis,  mate  made  from  the 

leaves  of,  184-185 
Indican,  urinary,  199,  258 
Indigo  series,  the,  197-201 
Indigo-blue,  formation  of,  200 
Indigo-carmine,  200 

Indol,  its  comijination  with  glycuronic 
acid,  107 
„      sources  of,  197 
,,      reactions  of,  198 
,,      fate  of,  in  the  body,  258 
Indoxyl  pigments,  258 
Indoxyl-sulphuric  acid,  199 
Inosit,  preparation  of,  108 

,,      reactions'  of,  109 
Intestine,   small,   hydrolysing  power  of 
secretion  of,  58-59 
,,         variable  reaction   of  its  con- 
tents, 63 
,,        its  inverting  action  on  cane- 
sugar,  110 
Inversion  of  laevulose,  106 

,,         of  cane-sugai',  110 
Invertebrates,  chitin  in  the  exoskeletons 
of,  87 
,,         tunicin  in  the  exoskeletons 

of,  101 
,,         haemoglobin   in  blood-plas- 
ma of,  217,  note 
,,         haemocyanin       in      blood- 
plasma  of,  230 
Invertin,  73 

Iron,  its  presence  in  haemoglobin,  224 
Iron-free  haematin,  238 
Isethionic  acid,  141 
Isinglass,  80,  note 
Isobutyric  acid,  118 
Isomerism,   physical    or  stereochemical, 

126,  128 
Isophenyl-ethylamin,  206 


276 


INDEX. 


JafFe's  test  for  indican,  200 

,,         ,,         skatoxyl,  203 
Jellies,  their  use  in  training  diets,  83 

KepMr,  preparation  of  from  mare's  milk, 

114 
Keratin,  composition  of,  86 
Keratinose,  86 

Ketones,  characteristics  of  the,  117 
Kola-nuts,  alkaloid  principle  of,  185 
Kreatin,  143 

,,         its  relation  to  kreatinin,  143 

,,         preparation  of,  144 

,,        its  relation  to  ui'ea,  162 
Kreatinin,  145 

,,         preparation  of,  146 
,,         reactions  of,  146-147 
Kresol,  195 

,,      reactions  of,  196 
Kresylsulphuric  acid,  195 
Kumys,  preparation  of  from  mare's  milk, 

114 
Kynureuic  acid,  192 

Lactalbumin,  23 
Lactic  acid  series,  the,  124-130 
Lactic  (hydroxy propionic)  acid,  124 
,,     its  presence  in  the  body,  125 
Lactic  fermentation  of  dextrose,  105 
Lactide,  how  formed,  128 
Lactoprotein,  24 

Lactose,  preparation  and  reactions  of,  113 
,,      lactic  fermentation  of,  114 
,,      its  incapability  of  assimilation, 
114 
Lsevulose,  synthesis  of,  101-102 

,,         chemistry  of,  106 
Lardacein,  or  amyloid  substance,  10 
,,  chemistry  of,  48 

,,  preparation  of,  49 

Laurie  or  laurostearic  acid,  1 19 
Lecithin,  133 

,,         a  constituent  of  egg-yolk,  26 
,,         preparation  of,  134 
,,         constitution  of,  135 
Lens,  crystalline,  globulin  of  the,  25 
Leprosy,  pigments  occurring  in,  255 
Leucin,  147 

,,        preparation  of,  148 
,,        a  result  of  decomposition  of  pro- 
teids,   40,  50,  79,  81,  84,  85, 
_  86,  147 
lieucomaines,  207 
Leukopsin,  264 

Liebig's  Extract  of  meat,  127,  178,  179 
Light,  its  bleaching  action  on   chloro- 

phanes,  262,  264 
Lignin,  99 
Ligroin,  156,  note 
Lipochrin   in   certain    retinal  epithelia, 

260,  262 
Lipochromes  or  luteins,  265 
'  Liquor    pancreaticus,'    its    amjdolytic 
power,  58 


'  Lithates,'  166 

'  Lithic  acid,'  166 

Liver,  formation  of  glycogen  in  the,  59 

"      conversion  of  glycogen  into  sugar 
in  the,  98 

,,      its  work  in  the  formation  of  urea, 
163,  171 

,,  ,,       in  the  formation  of  bile- 

pigments,  249 
Liver-sugar,  its  apparent  identity  with 

dextrose,  98 
Lobster,  chitin  obtained  from  the   exo- 

skeleton  of,  87 
Lupins,  xanthin  found  in,  175 
Lutein,  source  of,  266 
Luteins,  the,  265 

Lvmph,  dextrose  a  constituent  of,  102 
'  Lysatin,'  51,  161 

Malt-seedlings,  xanthin  present  in,  175 
Maltodextriu,  94 

Maltose,  its  conversion  into  dextrose,  57, 
59,  112 
,,         formation  of.  111 
Mantle  of    Tunicata,   tunicin    prepared 

from,  101 
Mantle-mucin  of  Helix  pomatia,  78 
Margaric  acid,  119 

Marrow  of  bones,  hemialbumin  in,  43 
Marsh-gas  fermentation  of  cellulose,  100 
Mate,  alkaloidal  principle  of,  184-185 
Meissner,  '  parapeptone  '  of,  36 

,,         his  researches  on  the  products 
of  digestion,  37,  38 
Melanin,  urinary,  256 
Melanins,  probable  differences  of,  262 
Melanogen,  256 
Metalbumin,  14 
Metapeptone,  Meissner's,  37 
Methsemoglobin,  preparation  of,  226 
,,     spectroscopy  of,  227 
,,     its  relation  to  oxyhsemoglobin,  229 
Methyl-glycin,  141 
Methyl-guanidinacetic  acid,  143 
Methyl-hydantoic  acid,  141 
Methyl-indol,  201 
Methylphenol,  196 
Micrococcus  ureffi,  158 
Micro-oi'ganisms,     their   appearance     in 
urine,  70,  71,  158 
,,  conversion  of  dextrose  by 

means  of,  105 
,,  hydration  of  urea  by,  158 

Milk,  preparation  of  casein  from,  20 
,,     clotting  of,  23 

,,     human,  and  of  cows  compared,  24 
„     conversion  of  lactose    into   lactic 

acid  in,  105 
„     varying  amounts  of  lactose  in,  113 
„     alcoholic  fermentation  of,  114 
Milk-sugar,  113 

Millon's  reagent  for  proteids,  7,  76 
Mucin,  reactions  of,  76 
,,       chief  sources  of,  77 


INDEX. 


277 


Murexid  test  for  uric  acid,  167 

Muscle,  ethyl-alcohol  obtained  from,  116 

,,     dead,  cause  of  acid  reaction  of,  128 

,,     living,  causes  of  acidity  of,  129 
Muscles,  presence    of  glycogen   in  the, 
95-96 

,,  ,,         of  inosit  in  the,  108 

,,  ,,        of  lactic  acid  in  the, 

125 

,,  ,,         of  sarcolactic   acid   in 

the,  126 

,,  „         of  hypoxanthin,  179 

Muscle-enzyme,  70 
Muscle-plasma,  clotting  of,  30,  70 
'  Myelin  forms'  of  lecithin,  134 
Myoglobulin,  31 
Myohajmatin,  235 
Myosin,  chemistry  of,  30 

,,         preparation  of,  31 
Myosin-ferment,  70 
Myosinogen,  31 
Myristic  acid,  119 

Nerves,   meduUated,    neurokeratin    ob- 
tained from,  87 
Neurin,  136 
Neurokeratin,  87 

,,        morphological  interest  of,  87 
Neutral  fats,  120 
Nitric-oxide  hsemoglobin,  222 
Nitrogen,  its  forms  in  proteid  matter,  52 
,,         its  presence  in  chondrin,  84 
,,         in  the  body,  asparagin  a  pos- 
sible source  of,  154 
, ,         in  urine,  method  of  determina- 
tion, 159 
„         its    mode   of    exit    from    the 
muscles,  160 
Nitrogenous  bodies  allied  to  proteids,  76 
,,  metabolism  lessened  by  gel- 

atin as  food,  82-83 
Nuclein,  preparation  and  properties  of, 

88 
Nucleo-albumins,  reactions  of,  89 


Olefines,  relation  of  oleic  acids  to  the,  120 
Oleic  acid,  a  constituent  of  human  fat, 

119,  120 
Olein  (tri-olein),  preparation  of,  122 
Orthodioxybenzol,  196 
-Osazones,  the,  101 

,,         formation  of  the,  102 
Ossein,  80 
Oxalic  acid  series,  the,  130 

,,         ,,         amido-acids  of  the,  151 
Oxaluricacid,  169,  171 
Oxybenzol,  193 

Oxychinolin-carboxilic  acid,  192 
Oxy-hsemoglobin,  preparation,  217 

„         difference  in  crystals  of  from 
different  sources,  218 

,,         spectra  of,  219 
Oyster,  presence  of  glycogen  in  the,  96 


Palm-oil,  palmitin  obtained  from,  121 
Palmitic  acid,  119,  121 
Palmitin  (tri-palmitin),  121 
Pancreas,  the  amylolytic  enzyme  of  the, 

57,  61 
Pancreatic  juice,    its   action  on  starch, 

111,  112 
Papain,  61 

,,         elastin  dissolved  by,  86 
Parabanic  acid,  169,  170 
Paradioxybenzol,  197 
Paraglobulin  (serum-globulin), chemistry 

of,  27 
Paramyosinogen,  31 
Parapeptone,  36,  37 
Paraxanthin,  177 

,,  isomer  of  theobromin,  177 

Penicillium,  effect  of  its  growth  on  gela- 
tin, 82 
,,      results  of  its  growth  in  ethy- 
lidene-lactic  acid,  128 
Pepsin,  preparation  oC,  59,  60 

,,       its    possible    combination    with 
hydrochloric  acid,  75 
Pepsinogen,  an  antecedent  of  pepsin,  61 
Peptones,  10,  36 

,,         retrospect  of  history  of,  44 
,,         preparation  of,  45 
,,        their  absorption  and  fate  in 
the  body,  46 
Petroleum- ether,  156,  note,  186 
Pettenkofer's  reaction  for  bile-acids,  213 
Phenol,  193 

,,         reactions  of,  195 
Phenylic-acid,  193 
Phenyl-glucosazone,  104 
Phenyl-hydrazin,    as    reagent     for     the 
sugars,  101 
,,     in  formation  of  osazones,  102 
,,     its  action  on  maltose,  112 
Phenyl-lactosazone,  114 
Phenyl-maltosazone,  preparation  of,  112 
Phenyl-sulphuric  acid,  194 
Phosphorus,  its  presence  in  casein,  20 
,,  a  constituent  of  mucin,  77 

,,  percentage  of  in  nuclein,  88 

,,  its  presence  in  protagon,137 

Phymatorhusin,  its  identity  with  mela- 
nin, 257 
Pialyn,  64 

Pigments  of  the  animal  body,  215 
,,         humus,  256 
,,         indoxyl-,  257 
,,         retinal,  260 
,,         of  urine,  251 
,,  ,,       asaffected  by  drugs,  260 

Piotrowski's  reaction  for  proteids,  7 
Piperazine,  140,  note 
Piria's  reaction  for  tyrosin,  191 
Plants,  occurrence  of  leucin  in,  147 

,,      proteid  metabolism  of,  52,  153 
'  Platelets,'  their  possible  connection  with 

clotting,  69 
Polarimeter,  forms  of,  103 


278 


INDEX. 


Propeptone,  43 
Propionic  acid,  117,  124 
Protagon,  137 
Protalbumose,  44 
Proteids,  5-53 

,,         composition  of,  5,  50-51 

,,         crystalline,  6 

,,         asii  of,  6 

,,         general  reactions  of,  7 

,,         classification  of,  9 

,,         coagulated,  10,  35 

,,         digestive  changes  of,  37,  38 

,,         duplexity  of  molecule  of,  38 

,,         their  decomposition  by  acids, 
40,  50 

,,        products  of  decomposition  of, 
49 

„         theories  of  the  constitution  of, 
51 
'  Protein  '  described  by  Mulder,  19 
Protogelatose,  82 
Pseudoxanthin,  207 
Ptomaines,  the,  204-207 

,,  their  similarity  to  vegetable 

alkaloids,  204,  205 
Ptyalin,  preparations  of,  56 

,,      its  action  on  starch,  57 
Purple,  visual-,  261,  264 
Pus-cells,  nuclein  prepared  from,  88 
Pus-corpuscles,  presence  of  glycogen  in, 

96 
Putrefactive  organisms,  action  on  cellu- 
lose of,  100 
Putrescin,  206 
Pyocyanin,  268 
Pyoxanthose,  268 
Pyrocatechin,  196 

Eennet,  use  of,  in  cheese-making,  65 
Rennin,  its  clotting  action  on  milk,  22, 
66 
,,      its  enzymic  nature,  65 
Rennin ogen,  66 

Retina,  pigments  of  the,  260-265 
Rhodophaiie,  261,  263 
Rhodopsin,  261,  264 
Rotation  of  light,  mode  of  measurement 
of,  103 

Saccharic  acid,  its  connection  with  gly- 
curonic  acid,  107 

Saccharose,  110 

Saliva,  ptyalin  a  constituent  of,  57 
,,       mucin  a  constituent  of,  76 
,,       its  action  on  starch-paste,  111 
,,       presence   of   sulpho-cyanates   in, 
163 

Salkowski-Ludwig  method,  estimation  of 
uric  acid  by  the,  167 

Sarcolactic,  or  paralactic  acid,  126,  127 

Sarkin,  180 

Sarkosin,  140 

Scherer's  test  for  nosit,  109 

Schiff's  reaction  for  uric  acid,  167 


Schultze's    reagent     for    cellulose,  100, 

note 
Schweizer's  reagent,  preparation  of,  99, 

note 
Seidel's  reaction  for  inosit,  110 
Serum  albumin,  chemistry  of,  12 

,,  preparation  of,  14 

Serum-casein,  27,  note 
Serum-globulin,  27 
Serum-lutein,  266 

Skatol,  its  combination  with  glycuronic 
acid,  107 
,,       j)reparations  of,  201 
,,       reactions  of,  202 
,,       occurrence  of,  in  a  vegetable  tis- 
sue, 203 
,,       compounds  of,  258 
Skatoxyl-pigments,  259 
Skatoxyl-sulphuric  acid,  202 
Snake's  eggs,  elastin-like  substance  in,  86 
Soaps,   formation   of,    with   stearic    and 
palmitic  acids,  119 
,,    composition  of,  124 
Soda,  sulphindigotate  of,  200 
Soluble  starch,  preparation  of,  92 
Spectrophotometers,  225 
Spectrophotometry,  224 
Spermaceti,  cetyl-alcohol  obtained  from, 

116 
Spermin,  139 

Spleen,  presence  of  inosit  in  the,  108 
,,     disintegration  of  red  corpuscles  in 
the,  250 
Starch,  hydrolysis  of,  by  ptyalin,  57 
,,  ,,         by    pancreatic    secre- 

tion, 57 
,,     sources  of,  91 
,,     molecule  of,  92 
,,     soluble,  92 

,,     digestion   of,  artificial  and  nor- 
mal, 94 
,,     its  conversion  into  sugar  in  the 
body,  94-95 
Starch  group  of  the  carbohydrates,  91 
Starch-paste,  action  of  saliva  on.  111 
Steaj'ic  acid,  119 

Stearin  (tri-stearin),  preparation  of,  121 
Strecker's  test  for  xanthin,  176 
Stroma  of  red  blood-corpuscles,  proteid 

constituent  of,  28 
Sub-maxillary  gland,  mucin  of  the,  77 
Succinic  acid,  131 
Sugar  in  blood,  determination  of,  8 
,,     conversion  of  hepatic  glycogen  in- 
to, 58,  98 
„     diabetic,  98 
Sugars,  the,  chemistry  of,  101 
,,         artificial,  102 
,,         discrimination  of,  102 
Sulphindigotate  of  soda,  200 
Sulpho-cyanic  acid,  its  formation  in  the 

body,  163 
Sulphur,  a  constituent  of  fibrin,  34 
,,         its  presence  in  lardacein,  48 


INDEX. 


279 


Sulphur,  its  presence  in  keratin,  86 

,,         a  constituent  of  cystin,  151 
Sulphuric  acid,  53 
Suprarenal  bodies,  pigment  of,  269 
Sweat,  presence  of  urea  in,  155 
Synovial  fluid,  nucleo-albumins  probably 

present  in,  90 
Syntonin,  chemistry  of,  16 
,,         preparation  of,  17 
,,         definition  of,  36,  note 

Taurin,  142 

Tauro-carbamic  acid,  143 
Taurocholic  acid,  preparation  of,  211 

,,  precipitation  of  proteids  by 

means  of,  212 
Tea,  traces  of  xanthin  present  in,  175 

,,     hypoxanthin  pi'esent  in,  179 
Teichmann's  crystals  (hsemin),  235 
Tendons,  mucin  of  the,  78 
Tetronerythrin,  sources  of,  267 
Theine,  its  relations  to  xanthin,  174,  184 
Theobroma  cacao,  its  alkaloidal  constit- 
uent, 184 
Theobromin,   its  relations    to  xanthin, 
173,  174,  184 
,,  isomer  of  paraxanthin,  177 

,,  an  excretionary  product  of 

plants,  185 
Theophyllin,    its   relations   to   xanthin, 

174,  178,  184 
Tinned  meats,  possible   development  of 

ptomaines  in,  205 
Torula  urese,  enzyme  developed  by,  70 
Touraco,  presence  of  copper  in  plumage 

of,  230 
Toxines,  205 
Trimethyl  vinyl-ammonium      hydroxide, 

136 
Tropseolins,    classification    of  acid-   and 

alkali-albumin  by  means  of  the,  18 
Trypsin,  its  action  on  fibrin,  34 

,,         its  action  on  proteids,  36,  38 
,,         preparations  of,  62 
Trypsinogen,  the  zymogen  of  trypsin,  64 
Tunicin,  101 
Turacin,  230 
Tyrein,  formation  of,  in  clotting  of  casein, 

22 
Tyrosin,    a  result   of  decomposition    of 
proteids,  40 
,,  a  product  of  decomposition  of 

mucin,  79 
,,  constitution  of,  189 

,,  preparation  of,  190 

,,  Hoffmann's  reaction  for,  191 

Umbilical  cord,  mucin  of  the,  79 
Urea,  155-164 

,,      average  daily  excretion  of,  155 

,,      preparation  of,  156 

,,      synthesis  of,  156,  159 

,,      nitrate  of,  156 

,,     oxalate  of,  157 


Urea,  detection  of,  in  solutions,  158 
,,     quantitative  determination  of,  159 
,,     its     probable     tissue-antecedents, 

160-162 
„     its  relations  to  uric  acid,  169-171 
Urea-ferment,  its  enzyniic  nature,  70,  71 
Ureas,  substituted,  163-164 
Uric  acid,  164-169 
„     salts  of,  166 
,,     preparation  of,  166 
,,     tests  for,  167 
,,     chemical  constitution  of,  168 
, ,     synthesis  of,  ]  69 
„     its  r-elations  to  urea,  169-171 
Urinary  melanin,  256 
.  Urine,  fermentative  changes  in,  70 

„      pathological  changes  in,  71,  102, 
108,  130,   203,  note,  206,   256, 
259 
,,      presence  of  kreatinin  in,  143 
,,      urea   the   chief  nitrogenous  con- 
stituent of,  155 
,,      determination  of  nitrogen  in,  159 
,,      sulpho-cyanates  present  in,  163 
„      phenyl-sulphuric  acid  in,  194 
,,      pyrocatechin  in,  196 
,,      pigments  of,  251-255 
Urobilin,  its  identity  with  hydrobiliru- 
bin,  246,  247,  248,  252 
,,         preparation  of,  252 
„         spectra  of,  253 
, ,         normal  and  febrile,  254 
,,         its  relation  to  other  pigment- 
ary substances,  253 
Urochrome,  254 
Uroerythrin,  255 
Urohfematin,  255 
UrohEematoporphyrin,  255 

Valeric  or  valerianic  acid,  118 
Van't  Hoff-Le  Bel  hypothesis  of  isomer- 
ism, 128 
Vegetable  alkaloids,  their  analogy  with 
ptomaines,  204,  205 
,,       tissues,  allantoin  found  in,  172 
„  ,,     xanthin  found  in,  175 

„  ,,     occurrence  of  hypoxanthin 

in,  179 
„  ,,    occurrence  of  guanin  in,  182 

,,  ,,     occurrence  of  skatol  in,  203 

Visual-purple,  261,  264 

„         action   of   light   and  reagents 
on,  264 
Vitellin,  chemistry  and  preparation  of,  26 

Water,  dependence  of  reactions  on  pres- 
ence of,  52 
,,      its  service  in  the  action  of  soluble 
ferments,  75 
Weidel's  reaction  for  xanthin,  176 
Weyl's  reaction  for  kreatinin,  147 

Xanthin  group,  the,  173-185 

Xanthin,  its  relationship  to  uric  acid,  174 


280 


INDEX. 


Xanthin,  preparation  of,  175 
,,        reactions  for,  176 
,,        derivatives  of,  184 
,,        physiological  action  of,  185 

Xaiithokreatiniii,  207 

Xanthophane,  261,  263 

Xanthoproteic  reaction  for  proteids,  7 

Xanthopsin,  264 

Yeast-cells,    early  observations  on,    72, 
73 


Yeast-cells,  nuclein  prepared  from,  88 

Zinc  lactate,  126 

,,     sarcolactate,  127 
Zymogen,  an  antecedent  of  the  enzymes, 
56 
,,         of  pepsin,  61 
,,         of  trypsin,  64 
Zymolysis,  53,  note 

,,  phenomena  of,  75 

,,  heat  phenomena  of,  75 


LIST  OF  AUTHORITIES  QUOTED. 


Those  mentioned  in  the  text  are  distinguished  by  an  asterisk. 


Abel,  Ladenburgu.,  139 

Almen,  195 

Andre,  Berthelot  et,  76 

An  rep,  von,  Weyl  u.,  222 

Araki,  228 

Argutinsky,  155 

Astaschewsky,  129 

Ayres,  Kiihne  and,  263,  266 

Baas,  189 

Baeyer,  136,  201 

Bagiusky,  117,  179,  181 

Barbieri,  Schulze  u.,  131,  154,  172,  188 
«Barth,  55 

Earth,  71 

Bary,  J.  de,  81,  84 

Banm,  187 
*Baiimann,  53,  194,  199 

Bauniann,  141,  150,  151,  189,  191,  193, 
194,  196,  197,  198,  260 

Bauniann  u.  Brieger,  196,  198,  200,  202 

Bauniann,  Christian!  u.,  195 

Baumann,  Goldmann  u.,  150 

Banmann  u.  Herter,  194,  196 

Baumann  u.  Hoppe-Seyler,  141 

Baumann  u.  v.  Mering,  141 

Baumann  u.  Preusse,  196,  197 

Baumann  u.  Tiemann,  200 
*Baumann,  Udranzsky  u.,  204 

Baumann,  Udranzsky  u.,  150,  206,  207 

Baumstark,  137,  255 

Bayer,  210 
*Beauraont,  37 
*Bechamp,  26,  51,  161 
*Behreud  u.  Roosen,  168 

Bein,  266 
*Bence- Jones,  39,  43 

Bensch,  105 
*Berard,  Corin  and,  11 

Berdez  u.  Nencki,  257 

Berlinerblau,  136 
*  Bernard,  59,  65,  95 

Bernard,  110,  111 
*Berthelot,  101 

Berthelot,  73,  88 

Berthelot  et  Andre,  76 


Berzelius,  74 

Bevan,  Cross  and,  99,  100 

Bidder  u.  Schmidt,  61 

Biedert,  24 

Biel,  24,  114 

Bimmermann,  59,  112 

Bizio,  96 

Bizzozero,  69 
*Blankenhorn,  137 

Blendermann,  192 

Bleunard,  86 

Boas,  66 

Bocklisch,  206 

Bodlander  u.  Traube,  9 

Boedeker,  108 

Boehm,  97,  136 
*Boehra  u.  Hoffmann,  97 

Boehm  u.  Hoffmann,  96 
*Bohr,  223 

Bohr,  222,  223 

Bohr  u.  Torup,  220 

Bokay,  89 
*Bokorny,  Low  u.,  52 

Bokorny,  Low  u.,  52 

Boll,  261 
*Bolton,  Chittenden  and,  12 

Borntrager,  Kiilz  u.,  98 

Bosshard,  Schulze  u.,  149,  154, 172,  182 

Bouchard,  207 

Bouehut,  Wurtz  et,  61 

Bourgeois,  Schiitzenberger  et,  81,  84 

Bourquelot,  59,  112 

Bourquelot,  Dastre  et,  59 

Bower,  100 

Brandl  u.  Pfeiffer,  258 
*Brieger,  137,  204,  205 

Brieger,  137,   191,  192,  193,  194,  196, 
197,  198,  202 

Brieger,   Baumann  u.,   196,  198,    200, 
202 

Brieger,  Stadthagen  u.,  207 
*Brown  and  Heron,  112 

Brown  and  Heron,  58,  59,  91,  111 

Brown  and  Morris,  92,  93,  94,  99,  107 

Brown,  Horace,  115 
*Briicke,  38,  59,  60,  67,  96 


282 


INDEX. 


Briicke,  34,  56 

Bruhns,  89,  181 

Brunton,  88 

Bruylants,  164 
*Buchanan,  67,  68 

Biitschli,  87 

Bufalini,  154 

Buliginsky,  194 
*Bunge,  7,  189 

Bunge,  88,  90,  100,  161,  171 
*Buusen,  159 

Buiisen  and  Roscoe,  225 

Burchard,  133 

Cahours,  Dumas  et,  26 

Camerer,  167 

Camjibell,  Heynsius  u.,  239,  245,  246 

Capranica,  145,  242,  263,  266 

Cash,  63 

Cazeneuve,  233 

Cazeneuve  et  Livon,  70 

Chaniewski,  122 
*Charcot,  139 

Chevalier,  87 

Chiari,  258 

Chittenden,  181 
*Chittendeu  and  Bolton,  12 

Chittenden  and  Goodwin,  32 

Chittenden  and  Hart,  85,  86 

Chittenden  and  Hartwell,  6,  26,  52 
»Chittenden,  Kuhne  u.,  5,  32,  44,  46 

Chittenden,  Kiihne  u.,  39,  42,  44,  47, 
60,  87 
*Chittenden  and  Painter,  25 

Chittenden  and  SoUey,  47,  82 

Chittenden  and  Whitehouse,  7 

Christiani  u.  Baumann,  195 

Church,  230 

Church,  Johnston  and,  185 

Claus,  173 
*Cloetta,  108 

Cohn,  191 
*Cohnheim,  57 

Cohnheim,  55 

Colasanti,  147 

Colasanti  and  Moscatelli,  126 

Commaille,  Millon  u. ,  21 

Coppola,  162 
*Corin  and  Berard,  11 
*Cornil,  48 
*Corvisart,  37 

Cramer,  97 

Cross  and  Bevan,  99,  100 

*.Danilewsky,  18,  58,  62 

Danilewsky,  P,  17,  31,  47,  56,  63 

Danilewsky  u.  Radenhausen,  21 

Dastre,  59,  98,  114 

Dastre  et  Bourquelot,  59 

Davidson  and  Dieterich,  61 

Demant,  31 
*Demarcay,  207,  208 
*Denis,  14,  30,  33 

Denis,  27 


Desmazieres,  72 

Dessaignes,  144 
*Diakouow,  137 

Diakonow,  136 

Dickinson,  47 

Dieterich,  Davidson  and,  61 

Disque,  247 

Donath,  71 
*Drechsel,  5,  6,  7,  50,  152,  161,  162,  210 

Drechsel,  5,  9,  43,  51,   144,  151,  181, 
213 
*Duboscq,  224 
*Dubruntaut,  111 

Dubrunfaut,  73 

Duggan,  Haycraft  and,  11 

Dumas  et  Cahours,  26 

Dunstan,  203 

Ebert,  120 

Ebstein,  239 

Ebstein  u.  Griitzner,  61 

Ebstein  u.  Miiller,  196 

Edkins,  Langley  and,  61 

Edlefsen,  28 

Ehrlich,  242 

Eichv.ald,  Kiihne  u.,  12 

Emich,  81,  82,  213 

Emich,  Maly  u.,  213 

Emmerling,  56 

Erlenmeyer,  66,  142 

Erlenmej'er  u.  Lipp,  189 

Erlenmeyer  u.  Schoffer,  85 

Erxleben,  72 

Esclier,  Hermann  u.,  83 

Etti,  243 

Etzinger,  80,  83 

Eugling,  23 

Eves,  59,  98 

Eves,  Langley  and,  57,  63 

Ewald,  80 

Ewald,  A.,  216,  218,  236 

Ewald  u.  Krukenberg,  182 

Ewald,  Kuhne  u.,  80,  87 

*Fano,  68 

Fano,  47,  69 

Feltz  et  Ritter,  249 

Fick,  110 

Filehne,  249 

Fileti,  201 
*Fiseher,  Emil,  101,  104,  168,  170,  177, 
178 

Fischer,  Emil,  175,  177,  179,  184,  202 

Fischer  u.  Passmore,  102 

Fischer  u.  Piloty,  107,  108 

Fitz,  268 

Flechsig,  100 

Flechsig,  Schulze  u.,  101 

Fleischer,  43 

Fleischl,  E.  von,  224 

Fordos,  268 

Franehimont,  101 

Fredericq,  133 
*Fremy,  26 


INDEX. 


283 


Frerichs  u.  Staedeler,  249 
Frey,  von,  126 
Friedberg,  66 
Friedreich  u.  Kekule,  48 
Friend,  Halliburton  and,  90 
Fubini,  Moleschott  u. ,  83 
Fudakowski,  106 
Fiihry-Snethlage,  28 
Funke,  133,  164 

Gabriel,  12 

Gaehtgens,  81 

Gaglio,  126 
*Ga,mgee,  67,  86,  137,  270 

Gamgee,  8,  27,  29,  67, 117, 127, 133,138, 
217,  224,  226,  228,  231,  234,  237, 262 
*Gautliier,  v.,  13 
*Gautier,  A.,  11,  204,  205,  207 

Gaiitier,  A.,  175 

Geoghegan,  188 

Gessard,  269 

Giacosa,  Nencki  u.,  196 
*Gilbert,  Lawes  and,  122 

Gilson,  134 

Girard,  268 

Glazebrook,  226 
«Gmelin,  208,  210 

Gmelin,  5 

Gmelin,  Tiedemann  u.,  242 

Goldmann  u.  Baumann,  150 

Goodwin,  Chittenden  and,  312 

Gorup-Besanez,  v.,  113,  189 
*Green,  34 

Green,  34,  68 

Green,  Lea  and,  68 

Grehant,  224 

Griessinayer,  92 
*Grimaux,  51 

Grimaux,  173 

Grohinann,  69 

Gruber,  Musculus  u.,  58,  111 

Grubert,  70 

Griitzner,  65 

Griltzner,  Ebstein  u.,  61 
*Gscheidlen,  125 

Gscheidlen,  155,  163 
*Guareschi,  147 
*Guareschi  e  Mosso,  204 

Haagen,  193 
*Haas,  12 
*Habermann,  50 

Habermann,  Hlasivvetz  u.,  149,  190 

Hallervorden,  163 
*Halliburton,  13,  14,  20,  28,  68 

Halliburton,  8,  23,  27,  30,  31,  43,  69, 
70,  90,  131,  196,  206,  217,  218,  228. 
229,  230,  238,  254,  267 

Halliburton  and  Friend,  90 
*Haniburger,  44 

Hamburger,  43 
*Hammarsten,  14,  21,  23,  28,  29,  30,  33, 
65,  66.  69,  78 

Hamnmrsten,  8,  12,  14,  20,   22,  24,  27, 


29,  30,  33,  42,  63,  65,  66,  77,  81,  90, 
105,  114,  213,  218,  229,  240,  267 

Harnack,  Sehmiedeberg  u.  136 

Hart,  Chittenden  and,  85,  86 

Hartweil,  Chittenden  and,  6,  26,  52 

Hasebroek,  34 

Haycraft,  47 

Haycraft  and  Duggan,  11 

Hayera,  69,  228  ' 
*Heidenhain,  64,  66 

Heidenhain,  56,  61,  63,  64,  129 
*Heintz,  65,  119,  125 

Heintz,  21,  65,  119 

Heller,  255 

Helwes,  61,  66 

Henneberg  u.  Stohmann,  101 
*Henninger,  45 

Henninger,  47 
*Hensen,  95 

Hermann,  83,  129 

Hermann,  L.,  222 

Hermann  u.  Escher,  83 

*  Heron,  Brown  and,  112 

Heron,  Brown  and,  58,  59,  91,  111 

Herrmann,  34,  71 

Herter,  Baumann  u.,  194,  196 
*Herth,  44,  45 

Herth,  42 

Herzfeld,  111 
*Hoschl,  48 

Hesse,  131,  195 

Heyl,  68 

Heynsius,  15,  28 

Heynsius  u.  Campbell,  239,  245,  246 
*Hilger,  86 

Hilger,  14,  109,  241 

Hirschler,  46 
*Hlasiwetz,  50 

Hlasiwetz  u.  Habermann,  149,  190 

Hogyes,  236 

Hoffmann,  A.,  189 

Hoffmann,  F.,  68 

*  Hoffmann,  F.  A.,  Boehm  u.,  97 
Hoffmann,  F.  A.,  Boehm  u.,  96 
Hoffmann,  H.,  61 

■  Hofmann,  K.  B.,  Ultzmannu.,  133, 164 
*Hofmeister,  45 
Hofmeister,  8,  12,  46,  60,  80,  81,   100, 

113,  151,  152,  192 
Hohlbeck,  134 
*Hoppe-Seyler,  5,  11,  17,  75,  134,  137, 
162,  209,  211,  216,  223,224,  231,  233, 
234,  248 
Hoppe-Seyler,  6,  7,  8,  9,  20,  21,  24,  26, 
27,  28,  53,   71,  73,  74,  81,   84,  87,  88, 
96,  100,  103,  1]0,  116,  134,  137,  146, 
155,  159, 171,  172,  211,  212, 215,  221, 
226,  228,  231,  232,  233,  234,  235,  237, 
238,  239,  241,  256 
Hoppe-Seyler,  G.,  194,  199,  202 
Hoppe-Seyler,  Baumann  u.,  141 
Horbaczewski,  81,  84,  85,  86,  143,  146, 
152,  168,  171 
*Hufner,  55,  58,  226 


284 


INDEX. 


Hiifner,  54,  71,  149,  217,  218,  220,  226, 

229 
Hiifner  u.  Kiilz,  230 
Hiifner  u.  Otto,  228 
Hundeshagen,  134 
Hiippe,  54 
Huppert,  143,  241 
Husemann,  205 

Jaarsveld  u.  Stockvis,  189 
Jacquemiu,  196 

Jaderholm,  222,  226,  228,  229,  231 
*Jatfe,  200,  247,  252 
Jaffe,  189,  192,  193,  199,  200 
Jatie,  Meyer  u.,  163 
Jaksch,  von,  70,  117,  158,  196,  257 
Jaquet,  7 
Jeanneret,  81 
Jernstrom,  79 
Johnson,  16 

Johnston  and  Church,  185 
Jolin,  220,  222 
Jong,  S.  de,  114 
Jonge,  D.  de,  116 

Katayama,  222 

Kekule,  Friedreich  u.,  48 

Kieseritzky,  18 

Kistiakowsky,  34 
*Kjeldahl,  159 

Kjeldahl,  71 

Klemptner,  70 

King,  47,  82 

Knieriem,  v.,  153,  154 

Kobert,  185 

Kochs,  195 

Koebner,  111 

Konig,  113,  131 

Koster    22 
*Kossel,'45,  89,  178,  181,  182 

Kossel,  88,  89,  90,   176,  177,  179,  181, 
182,  184 

Kostjurin,  49 

Koukol-Yasnopolsky,  198 

Krannhals,  114 

Kratschmer,  Seegen  u.,  59 

Kratter,  120 

Krause,  180 

Krawkow,  55,  57 

Kretschy,  193 

Kreusler,  Ritthausen  u.,  153 

Krohn,  264 

Kriiger,  A.,  6,  68 

Kriiger,  M.,  181 
*Krukenberg,  4,  48,  147,  268,  269 

Krukenberg,  7,   84,  86,   87,  145,  147, 
195,  230,  243,  267 

Krukenberg,  Ewald  u.,  182 

Krukenberg  u.  Wagner,  178 

Kriiss,  G.  u.  H.,  224,  226,  258 

Kugler,  70 
*Kiihne,  17,  37,  38,  39,  40,  41,  42,  48, 
55,  62,  86, 109,  217,  232,  249,  261,  263 

Kiihne,  8,  17,  21,  27,  28,  30,  34,  36,  39 


44,  45,  47,  53,  55,  57,  63,  64,  74,  96, 

198,  215,  262,  263 
Kiihne  and  Ayres,  263,  266 
*Kiihue  u.  Chittenden,  5,  32,  44,  46 
Kiihne  u.  Chittenden,  39,  42,  44,  47, 

60,  87 
Kiihne  u.  Eichwald,  12 
Kiihne  u.  Ewald,- 80,  87 
Kiihne  u.  Eudneff,  49 
Kiihne  u.  Sewall,  182 
*Kulz,  97,  130     . 
Kulz,  59,  97,  98,  108,  111,  150,  218,  221 
Kiilz  u.  Borntrager,  98 
Kiilz,  Hiifner  u.,  230 
Kiissner,  191 
Kunkel,  75,  222 
Kunz,  268 

Ladenburg,  206 

Ladenburg  u.  Abel,  139 

Lahorio,  236 

Lailler,  71 

Laker,  69 

Lambling,  224 

Landolt,  103,  195 
*Landwehr,  79,  95,  97 

Landwehr,  14,  30,  77,  95,  97 

LangendorfF,  98 

Langgaard,  24 

Langley,  57,  61,  66,  76 

Langley  and  Edkins,  61 

Langley  and  Eves,  57,  63 

Laptschinsky,  11,  25 

Latchenberger,  249 

Latschinoff,  209 
*Latour,  Cagniai'd  de,  72 
*Lawes  and  Gilbert,  122 

Lea,  63,  71,  95,  148,  153,  158 

Lea  and  Green,  68 

Le  Bel,  128 

Ledderho^e,  87 

Legal,  198 

Lehnmnn,  28,  36,  37,  83,  120 

Lepine,  75 
*Leube,  127 

Leube,  70,  110,  111 

Leube,  Salkowski  u.,  43,155,  171,  188, 
194,  240 
*Leuwenhoek,  72 

Levy,  235 

Lewkowitsch,  129,  149 

Leydig,  261 
*Lieberkiihn,  19 

Lieberniann,  79,  89,  243,  248 
*Liebig,  16,  72,  73,  75,  128,  129,  159, 
192 

Liebig,  73,  127,  131 
*Liebreich,  137 

Liebreich,  137 

Limbourg,  34 

Limpricht,  98 
*Lindberger,  64 

Lindberger,  63,  213 

Lindet,  94 


INDEX. 


285 


♦Lindwall,  87 

Lindwall,  86 

Lipp,  198 

Lipp,  Erlenmeyer  u.,  189 

Lippmanu,  149,  191 

Lister,  105 

Livon,  Cazeneuve  et,  70 

Lobisch,  78 
*Lbw,  45,  51,  55 

Low,  51,  60,  62,  71,  88,  161,  181 
*Low  u.  Bokorny,  52 

Low  u.  Bokorny,  52 
*Lowit,  08 

Lowit,  69 

Longo,  von,  131,  154 
*Lossen,  51 

Lossen,  161 

Lossnitzer,  58 
*Lubavin,  21 

Lubavin,  90 

Ludwig,  29,  69,  152,  167,  171 

Liicke,  268 

Lundberg,  22,  23 

McKendrick,  134 

Mach,  von,  181 
*MacMunn,  250,  253,  255 

MacMunn,  213,  233,  235,  237,  238,  247, 
253,  254,  255,  258,  267,  269 

Majert  u.  Schmidt,  139 

Makris,  25 

Malassez,  224 
*Maly,  45,   61,  75,  125,  126,  240,   244, 
247,  254,  266 

Malv,  56,  57,  105,  211,  239,  240,  244, 
246,  247,  267 

Maly  u.  Einich,  213 
* Mantegazza,  67,  68 
*Maquenne,  108 

Marouse,  126,  163 

Marino-Zuco,  135 

Marme,  108 

Mnrpmann,  105 

Marshall,  218,  226 

Martin,  61 

Masius,  Vanlair  u.,  247 

Mathieu  et  Urbain,  133 

Mauthner,  149,  151,  191 

Mauthner  u.  Suida,  140 

Maydl,  98 
*  Mayer,  Ad.,  74 

Mayer,  Ad.,  61,  66,  71,  75 

Mayet,  217 

Mays,  63 
«Medicus,  168 

Mehn,  253 

Meissl,  106,  111 

Meissl  u.  Strohmer,  122 
*Meissner,  36,  37,  38,  39,  41,  42,  43 

Meisaner  u.  Shepard,  189 

Merejkowski,  267 
*"iVrering,  von,  84 

Mering,  von,  58,  84,  107,  111,  115 

Mering,  von,  Baumann  u.,  141 


Mering,  von,  Musculus  u.,  58,  59,  96, 
98,  111,  112 

Master,  203,  259 

Meyer,  105 

Meyer  u.  Jaffe,  163 

Meyer,  Musculus  u.,  93,  106 

Meyer,  Schniiedeberg  u.,  107 
*Mialhe,  37,  56 

Mialhe,  36 

Michael,  173 

Michailow,  28 

Michelson,  70 

Miescher,  88 

Miller,  91,  103,  115,  128 

Millon  u.  Commaille,  21 

Minkowski,  126,  163 

Minkowski  u.  Naunyu,  250 

Mii|uel,  70 

Miura,  257 

Modrzejewski,  48 
*Morner,  16,  17,  84 

Morner,  17,  18,  84,  256 

Moleschott,  242 

Moleschott  u.  Fubini,  83 

Morochowetz,  84 

Morris,  Brown  and,  92,  93,  94,  99,  107 

Moscatelli,  Colasanti  and,  126 

Mosso,  Guareschi  e,  204 
*Mulder,  19,  37 

Miiller,  H.,  261 
*Muller,  W.,  108,  138 

Miiller,  W.,  70 

Miiller,  Fr.,  199 

Miiller,  Ebstein  u.,  196 

Munk,  122,  163,  260 

Muntz,  56 
*Musculus,  71 

Musculus,  158 

Musculus  u.  Gruber,  58,  111 

Musculus  u.  Meyer,  93,  106 

Musculus  u.  von  Mering,  58,  59,  96,  98, 
111,  112 

Musso,  75 

Mylius,  209,  211,  214 

*Na,geli,  von,  73,  74 

Nageli,  von,  75 
*iSrasse,  32 

Nasse,  50,  59,  96,  97 
*Naunyn,  249 

Naunyn,  96,  249 

Naunjm,  Minkowski  u.,  250 

Nebelthau,  126 
*Nencki,  206 

Nencki,  81,  198,  199,  201,  258 

Nencki,  Berdez  u.,  257 

Nencki  u.  Giacosa,  196 

Nencki  u.  Rotschy,  237 

Nencki,  Schultzen  u.,  191 
*Nencki  u.  Sieber,  248 

Nencki  u.  Sieber,  216,  234,  237,  257 

Nessler,  66 

Neubauer,  175 
*Neubauer  u.  Vogel,  159 


286 


INDEX. 


Neubauer  u.  Vogel,  104,  107,  113,  117, 
130,  146,  155,  171,  172,  173,  194,  200, 
206,  215,  226,  243,  251,  252 

Neumann,  69 
*Neumeister,  26 

Neumeister,  24,  34,  44,  47 

iSTiggeler,  193 

Nikoljukin,  25 

Noorden,  von,  226 

Nussbaum,  223 

Obolensky,  14,  77 
Odo'inatt,  193 
Ord,  258 
Ortweiler,  199 
*0'Sullivan,  94,  111 
Otto,  34,  46,  47,  203,   205,  206,  217, 
226,  229,  259 
Otto,  Hlifner  u.,  228 

*Paiikull,  77 

PaijkuU,  77,  90 
*Painter,  Chittenden  and,  25 

Palm,  24 

Panonnow,  97,  98 
*Panum,  206 

Panum,  27 

Parens,  138 

Parke,  212 
*Pasehutiu,  58,  65 

Paschutin,  111 

Passmore,  Fischer  u. ,  102 
*Pasteur,  72 

Pasteur,  70,  123,  158 

Pecile,  182 

Pekelharing,  47 

Pelouze,  224 

Petit,  61 

Petri,  84 
*Pettenkofer,  214 

Pfeitfer,  20 

Pfeiffer,  Brand!  u.,  256 
*Pnuger,  52,  159,  162 

Philips,  59,  112 

Piloty,  Fischer  u.,  107,  108 

Piria,  154 

Piutti,  154 
*P16sz,  256 

Plosz,  14,  32,  34,  88,  90 

Plugge,  195 

Podolinski,  64 

Podwj^ssozky,  61 

Poehl,  139 

Poggiale,  29 

Pohl,  89 

Polak,  61 

Pollitzer,  47 

Popoff",  100 

Pouchet,  178 

Poulton,  116 

Preusse,  196,  197 

Preusse,  Baumann  u.,  196,  197 
*Preyer,  32 

Preyer,  96,  221,  234 


Quincke,  260 
Quinquaud,  224 

Kadenhausen,  Danilewski  u.,  21 

Radziejewski,  197 

Radziejewski  u.  Salkowski,  152 

Rajewski,  116 

Ranke,  129 
*Raoult,  92 
*Rauschenbach,  68 

Rauschenbach,  69 
*Reauniur,  37 

Rechenberg,  76 
*Reinke,  4 

Reissert,  157 

Reymond,  Du  Bois,  128 

Rindfleisch,  69 

Ringer,  22,  66 

Risler,  Schtitzenberger  et,  224 
*Ritter,  51,  161 

Ritter,  Feltz  et,  249 

Ritthausen  n.  Kreusler,  153 
*Roberts,  58,  66 

Robin,  135      " 

Rodewald  u.  ToUens,  113 

Rohmann,  44,  154,  191 
*Rollett,  14 

Rollett,  15 
=*Roosen,  Behrend  u.,  168 

Roscoe,  Bunsen  and,  225 

Rosenberg,  18 

Rossbuch,  185 

Roster,  .163 

Rotschy,  Nencki  u.,  237 

Rubner,  76,  122 

Eudneff,  Kiihne  u.,  49 

Sachsse,  92 
*Salkowski,  E.,  43,  127,  162 

Salkowski,  E.,  34,  42,  54,  71,  131,  141, 
143,  145,  147,  148,  171, 172,  179,  188, 
194,  195,  198,  203,  222,  238,  239,  253, 
256 

Salkowski,  E.  u.  H.,  188,  191,  201,  203 

Salkowski  u.  Leube,  43,  155,  171,  188, 
194,  240 

Salkowski,  Radziejewski  u.,  152 

Salomon,  G.,  176,  177,  178,  179,  180, 
181,  182 

Salomon,  W.,  163,  189 

Samson-Himmelstjerna,  68 

Sander,  17 
*Schafer,  14 

Schafer,  101,  250 

Schalfejew,  236,  237 

Schefter,  61 

Schenk,  213 
*Scherer,  14,  108 
*Schiff,  38 

Schiff,  158 

Schiffer,  141 

Schimmelbusch,  69 

Schindler,  89,  181 

Schmidt,  Albr.,  Majert  u.,  139 


INDEX. 


287 


*Schmi(it,  Alexander,  14,  28,  30,  34,  67, 
68,  70 
Schmidt,  Alexander,  22,  27,  29,  54,  65, 
138 
*Sclimidt,  Aug.,  55 
Schmidt,  C,  48 
Schmidt,  C,  Bidder  u.,  61 
Schmidt-Mulheim,  36,  42,  46,  47,   63, 
132,  133 
*Schmiedeberg,  162,  189 
Schmiedeberg,  189,  196,  197,  204 
Schmiedeberg  u.  Harnack,  136 
Schmiedeberg  u.  Schultzen,  192 
Schmiedeberg  u.  Meyer,  107 
Schoffer,  Erlenmeyer  u.,  85 
Schotten,  191,  209 
Schreiner,  139 
*Schroder,  von,  163 
Schroder,  von,  171 
*Schrotter,  52 

Schulz,  101 
*Schulz,  0.,  204 
Schulze,  E.,  50,  149,  154,  190,  191 
Schulze  u.  Barbieri,  131,  154,  172,  188 
Schulze  u.  Bosshard,  149,  154,  172,  182 
Schulze  u.  Flechsig,  101 
*Schultzen,  141 
Schultzen,  141 
Schultzen  u.  Nencki,  191 
Schultzen,  Schmiedeberg  u.,  192 
Schumberg,  66 
*Schiitzenberger,  39,  41,  50 
Schiitzenberger,  51 
Schiitzenberger  et  Bourgeois,  81,  84 
Schiitzenberger  et  Risler,  224 
Schwalbe,  23 
*Schwann,  72 
Schweder,  82 
Sczelkow,  226 
Sebelien,  23 
Secretan,  201 
Seegen,  8,  59,  98 
Seegen  u.  Kratschmer,  59 
Sellden,  61 
*Selmi,  204 
Selmi,  21 
Sembritzky,  24 
Senator,  28,  171,  200 
*Setscheno\v,  133 
Setschenow,  221 
Sey  berth,  142 
Sewall,  Klihne  u.,  182 
Sliepard,  Meissner  u.,  189 
Sieber,  262 
*Sieber,  Nencki  u.,  248 
Sieber,  Nencki  u.,  216,  234,  237,  257 
Siegfried,  51,  129 
Simon,  24 
Smith,  H.  E.,  87 
Solley,  Chittenden  and,  47,  82 
Sotnitschewsky,  135 
*Soxhlet,  23 
Soxhlet,   19,  22,  23,  66,  103,  106,  111, 
112,  113 


Soyka,  16,  18,  19 
*Spallanzani,  37 

Spiro,  126 
*Stadelmann,  130 

Stadelmann,  61,  163,  249 

Stadthagen,  179,  181 

Stadthagen  u.  Brieger,  207 
*Stadeler,  243,  244 

Stadeler,  51,  175,  240 

Stadeler,  Frerichs  u.,  249 

Stahl,  72 
*Starke,  12,  13 

Starke,  11,  12,  13,  14 
*Stas-Otto,  205 

Steinbrligge,  87 

Steiner,  249 

Stern,  250 
*Stevens,  37 

Stbckly,  201 

Stohmann,  76 

Stohmann,  Hennebergu. ,  101 
*Stokes,  231,  233 

Stokvis,  243,  246,  259 

Stokvis,  Jaarsveld  u.,  189 

Strassburg,  214,  223 
*Straub,  44 

Straub,  42 
*Strecker,  135,  208 

Strecker,  182 

Strohnier,  Meissl  u.,  122 

Struve,  21,  114 

Suida,  Mauthner  u.,  140 
*Sundberg,  60 

Sundwik,  87 

Szabo,  63 

Tappeiner,  51,  100,  161,  188 
*Tarchanoff,  249 

Tatarinoff,  47,  81 

Teichmann,  235 

Thierfelder,  107 

Thoiss,  181 
*Thudichuni,  106,  254,  256 

Thudichum,  177,  244 

Tiedemann  u.  Gmelin,  242 

Tieghem,  van,  70,  100,  158 

Tiemann,  Baumann  u.,  200 

Tollens,  91,  104 

ToUens,  Rodewald  u.,  113 

Tolmatscheff,  132,  133 

Torup,  223 

Tornp,  Bohr  u.,  220 

Traube,  74 

Traube,  Bodlanderu.,  9 

Tscherwinsky,  122 

*Udranskv,  214 

Udransky,  191,  195,  196,  198,  213,  214, 
251,  256 
*Udransky  u.  Baumann,  204 

Udransky  u.  Baumann,  150,  206,  207 

Uffelmann,  82,  127 

Ultzmann  u.  K.  B.  Hofmann,  133,  164 


288 


INDEX. 


Umbacli,  260 

Urbain,  Matliieu  et,  133 

*Va]enciennes,  26 

Vanlair  u,  Masius,  247 
*Vaii't  HofF,  128 

Van't  HofF-Le  Bel,  128 

Velden,  v.  d.,  63 

Vella,  59,  111 
*Vierordt,  225 

Vierordt,  225,  226,  243,  246,  247,  251, 
254,  258 

Vines,  6,  36,  153 
*Virchow,  248 

Virchow,  36,  48,  151,  182,  239 
*Vogel,  Neubauer  u.,  159 

Vogel,  Neubauer  u.,  104, 107,  113,  117, 
130,  146,  155,  171,  172,  173,  194, 
200,  206,  215,  226,  243,  251,  252 

Vohl,  108,  109 
*Voit,  83,  122,  154 

Voit,  83,  122,  143,  145,  154,  185 

Volhard,  143,  168 

Vossius,  243,  249 

Walchli,  79,  85,  199 
Wagner,  Kvukenberg  u.,  178 
Warren,  129 
Wasilewski,  61 
Wedenski,  95,  256 
Weidel,  178 

Weiske,  80,  81,  101,  154,  188 
*Weiske  and  Wildt,  154 
Weiss,  64 


Welzel,  222 

Wenz,  8,  45 

Werigo,  14 

Werther  129 

Wevl,  6^  26,  31,  32,  48,  81,  154,  191, 
198,  199 

Weyl  u.  von  Anrep,  222 

Weyl  u.  Zeitler,  129 

Whitehouse,  Chittenden  and,  7 
*  Wildt,  Weiske  and,  154 

Wislicenus,  127,  129 
*Wittich,  von,  58 

Wittich,  von,  55,  74 
*Wohler,  156,  186 

Wohl,  95,  110 

Wolifberg,  223 

Wooldridge,  28,  29 
*Wooldridge,  69 

Worm-Miiller,  88,  89,  147 

Wurm,  267 

Wurster,  191 
*Wurtz,  55,  136 

Wurtz,  61,  75 

Wurtz  et  Boucliut,  61 

Zahn,  23 
Zaleski,  223,  250 
Zeitler,  Weyl  u.,  129 
Zeller,  256 
Zenker,  138 
Zillner,  120 
ZinofFsky,  217,  218 
Zuntz,  133,  221 
Zweifel,  6Q 


Jtist  Ready. 

Text-Book  of  Embryology:  Man  and  Mammals. 

By  DK.  OSCAR  HERTWIG, 

PROFESSOR   OF   ANATOMY   AND    COMPARATIVE   ANATOMY,    DIRECTOR  OF 
THE   II.    ANATOMICAL   INSTITUTE,    UNIVERSITY    OF   BERLIN. 

TRANSLATED  AND  EDITED  FROM  THE  THIRD  GERMAN   EDITION 

BY 

EDWARD   LAURENS   MARK,    Ph.D., 

HERSEY   PROFESSOR  OF   ANATOMY,    HARVARD  UNIVERSITY. 

Fully  Illustrated.    Demy  8vo. 

"The  Embryology  of  Animals,  although  one  of  the  youngest  shoots  of  morphological 
research,  has,  nevertheless,  grown  up  in  the  course  of  sixty  years,  along  M'ith  the  cell- 
doctrine  and  that  of  the  tissues,  to  a  vigorous  and  stately  tree.  The  comprehension  of  the 
structure  of  organisms  has  been  extended  in  a  high  degree  by  numerous  developmental 
investigations.  The  study  nf  the  human  horly  has  also  derived  great  advantage  from  tlie 
same.  In  the  newer  anatomical  text-books  Embryology  is  receiving  more  and  more  atten- 
tion in  the  description  of  the  separate  systoms  of  organs.  To  what  extent  man\'  things 
mn}'  be  more  clearly  and  attractively  described  in  this^  manner  is  best  shown  b}-  a  compari- 
son of  tire  descriptions  of  brain,  eye,  heart,  etc,  in  the  older  and  the  more  recent  anatomi- 
cal text-books. 

"Although  it  is  generally  recognised  that  Emh'yology  constitutes  'a  foundation  of  our 
comprehension  of  organic  forms,'  nevertheless  the  attention  which  its  importance  warrants 
is  not  yet  given  to  it;  it  is  especial!}'  true  that  it  has  not  become  as  extensively  as  it  should 
be  a  component  of  well-rounded  medical  and  natural-historj'  instruction,  to  which  it  is 
indispensable.  ...  I  have  in  the  present  text-book  placed  the  comjmrative  method  of 
invesli(jntion  in  the  foreground."  —  From  the  Author's  Preface. 

"  The  rapidly  increasing  recognition  of  the  importance  of  Embryology  in  all  mor- 
phological studies  makes  it  desirable  that  the  most  valuable  text-books  upon  the  subject,  in 
wliatever  language,  be  made  available  for  those  who  are  beginning  its  study.  Although 
the  English-reading  student  alreadj'  has  at  command  a  number  ot  text-books  upon  this 
subject,  it  is  evident  to  any  one  familiar  with  Hertwig's  Lehrbuch  der  Entwichlungsge- 
schiclite  des  Menschen  unci  der  Wirbelthiere  that  this  work  covers  the  field  of  Vertebrate 
Embryology  in  a  more  complete  and  satisfactory  way  than  any  book  heretofore  published 
in  English."  —  From  the  Translator's  Preface. 

In  the  Press. 

Text-Book  of  Embryology  :  Invertebrates. 


By   DRS.  KORSCHELT   and   HEIDER, 

PRIVATDOCENTEN,    university    of   BERLIN. 
TRAN.SLATED   AND   EDITED   BY 

EDWARD   LAURENS   MARK,  Ph.D., 

HERSEY   PROFESSOR  OF    ANATOMY,    HARVARD   UNIVERSITY, 
AND 

WILLIAM   McMICHAEL  WOOD  WORTH,  Ph.D., 

INSTRUCTOR   IN   MICROSCOPICAL  ANATOMY,    HARVARD    UNIVERSITY. 

Fully  Illustrated.    Demy  8vo. 


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Jvtst  Published,  -with  j8^  Illustrations.     8vo.    $^-30. 

TEXT-BOOK  OF  COMPARATIVE  ANATOMY, 

By  dr.  ARNOLD    LANG, 

PROFESSOR    OF    ZOOLOGY    IN    THE    UNIVERSITY     OF     ZURICH  ;     FORMERLY 
RITTER  PROFESSOR   OF   PHYLOGENY   IN    THE    UNIVERSITY   OF   JENA. 

With  Pf'eface  to  the  English   Translatioft 
By   professor   DR.  ERNST    HAECKEL,  F.R.S., 

DIRECTOR   OF   THE    ZOOLOGICAL    INSTITUTE    IN   JENA. 

Translated  into  English  by 
HENRY   M.  BERNARD,  M.A.  (Cantab.),  and   MATILDA   BERNARD. 

^art  I. 

Complete,  with  Index  and  383  illustrations.     8vo.     I5.50. 


This  translation  of  the  first  volume  of  Professor  Lang's  Lehrbuch  der 
Vergleichende  Anatomie  may  be  considered  as  a  second  edition  of  the 
oi;iginal  work.  Professor  Lang  kindly  placed  at  our  disposal  his  notes, 
collected  for  the  purposes  of  emendation  and  expansion,  and  they  have 
been  duly  incorporated  in  the  text.  —  From  the  Translator'' s  Preface. 

Professor  Lang  has  here  successfully  carried  out  the  very  difficult 
task  of  selecting  the  most  important  results  from  the  bewildering  mass 
of  new  material  afforded  by  the  extensive  researches  of  the  last  decades, 
and  of  combining  them  with  great  judgment.  Besides  this  he  has,  more 
than  any  former  writer,  utilized  the  comparative  history  of  development 
in  explaining  the  structure  of  the  animal  body,  and  has  endeavored 
always  to  give  the  phylogenetic  significance  of  ontogenetic  facts.  Lastly, 
he  has,  by  the  clear  systematic  reviews  of  the  various  classes  and  orders 
which  precede  the  anatomical  account  of  each  race,  further  facili- 
tated the  phylogenetic  comprehension  of  complicated  morphological 
problems,  his  wisely  chosen  and  carefully  executed  illustrations  assist- 
ing materially  in  this  result.  It  is  therefore  with  great  pleasure  that  I 
commend  this  book  to  the  English  student,  in  the  hope  that  the  English 
translation  will  promote  to  as  great  an  extent  as  the  German  original  the 
wider  study  and  better  comprehension  of  animal  morphology,  and 
will  attract  new  students  to  this  noble  science.  ■ —  From  Professor  Haec- 
kel's  Preface. 

.     MACMILLAN  &  CO.,  112  Fourth  Avenue,  New  York. 


WORKS   BY   MICHAEL   FOSTER, 

M.A.,    M.D.,    LL.D.,    F.R.S., 

PROFESSOR    OF    PHYSIOLOGY    IN   THE    UNIVERSITY    OF   CAMBRIDGE,    AND    FELLOW 
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A  Text-Book  of  Physiology. 

With  illustrations.     Fifth  Edition.     Largely  revised. 

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The  Tissues  and  Mechanisms  of  Reproduction.  8vo.  $2  00. 
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Body.     By  A.  Sheridan  Lea,  M.A.,  Sc.D.,  F.R.S. 

"The  present  edition  is  more  than  largely  revised.  Much  of  it  is  re-written, 
and  it  is  brought  quite  abreast  with  the  latest  wave  of  progress  of  ijhysiological 
science.  A  chief  merit  of  this  work  is  its  judicial  temper,  a  strict  sifting  of  fact 
from  fiction,  the  discouragement  of  conclusions  based  on  inadecjuate  data,  and  small 
liking  shown  toward  fanciful  though  fascinating  hyj^otheses,  and  the  avowal  that  to 
many  questions,  and  some  of  foremost  interest  and  moment,  no  satisfying  answers 
can  yet  be  given."  —  Neiu  England  Medical  Journal. 

"  It  is  in  all  respects  an  ideal  text-book.  It  is  only  the  physiologist,  who  has 
devoted  time  to  the  study  of  some  branch  of  the  great  science,  who  can  read  between 
the  lines  of  this  wonderfullv  generalized  account,  and  can  see  upon  what  an  intimate 
and  extensive  knowledge  these  generalizations  are  founded.  It  is  only  the  teacher 
who  can  appreciate  the  judicious  balancing  of  evidence  and  the  power  of  presenting 
the  conclusions  in  such  clear  and  lucid  forms.  But  by  every  one  the  rare  modesty 
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embryology."  —  Academy. 

A  Course  of  Elementary  Practical 
Physiology. 

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elementary  experimental  physiology.  Its  chief  utility,  however,  will  be  to  the 
intellige;it  student,  who  armed  with  a  dissecting  case,  a  microscope,  and  the  book, 
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profitable."  —  Medical  Record. 


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sity of  Jena."  Issued  as  the  Ninth  Edition  of  Edward  Oscar 
Schmidt's  "  Handbook  of  Comparative  x\natomy."  Translated 
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QP34 


1891 


